Rat Liver Grafts Research Articles

Statement of Retraction: Liver X Receptors Activation Attenuates Ischemia Reperfusion Injury of Liver Graft in Rats

Statement of Retraction: Liver X Receptors Activation Attenuates Ischemia Reperfusion Injury of Liver Graft in Rats

Exploration of Optimal pH in Hypothermic Machine Perfusion for Rat Liver Grafts Retrieved after Circulatory Death.

Ex vivo hypothermic machine perfusion (HMP) is a strategy for controlling ischemia-reperfusion injury in donation after circulatory death (DCD) liver transplantation. The pH of blood increases with a decrease in temperature and water dissociation, leading to a decrease in [H+]. This study aimed to verify the optimal pH of HMP for DCD livers. Rat livers were retrieved 30 min post-cardiac arrest and subjected to 3-h cold storage (CS) in UW solution (CS group) or HMP with UW-gluconate solution (machine perfusion [MP] group) of pH 7.4 (original), 7.6, 7.8, and 8.0 (MP-pH 7.6, 7.8, 8.0 groups, respectively) at 7-10 °C. The livers were subjected to normothermic perfusion to simulate reperfusion after HMP. All HMP groups showed greater graft protection compared to the CS group due to the lower levels of liver enzymes in the former. The MP-pH 7.8 group showed significant protection, evidenced by bile production, diminished tissue injury, and reduced flavin mononucleotide leakage, and further analysis by scanning electron microscopy revealed a well-preserved structure of the mitochondrial cristae. Therefore, the optimum pH of 7.8 enhanced the protective effect of HMP by preserving the structure and function of the mitochondria, leading to reduced reperfusion injury in the DCD liver.

AAV9-mediated gene delivery to liver grafts during static cold storage in a rat liver transplant model.

Recombinant adeno-associated virus (rAAV) is a novel strategy used clinically for gene delivery, but has not been characterized in the context of organ transplantation. We sought to determine the efficacy of rAAV-mediated gene delivery during static cold storage (SCS) prior to liver transplantation. A triple-plasmid transfection protocol was used to produce rAAV subtype-9 vectors containing firefly luciferase genomes in HEK293 cells. Lewis rat liver grafts were flushed and stored in cold HTK solution. Three experimental groups received rAAV at different doses, administered via the portal vein as a bolus during SCS. A control group did not receive rAAV (N = 2). Recipients then underwent syngeneic liver transplantation. Bioluminescence imaging to quantify in vivo luciferase expression was performed on post-operative days 7, 14, 28, and 56. Control animals demonstrated no bioluminescent activity, while animals receiving rAAV-treated livers had increasing bioluminescence, peaking at four weeks but sustained to the eight-week endpoint. This result was confirmed by experimental endpoint tissue luciferase activity assay. rAAV mediates gene transduction in liver grafts when administered during SCS and has potential for gene therapy applications in solid organ transplantation.

Hypothermic Oxygenated Machine Perfusion Promotes Mitophagy Flux against Hypoxia-Ischemic Injury in Rat DCD Liver

Hypothermic oxygenated machine perfusion (HOPE) can enhance organ preservation and protect mitochondria from hypoxia-ischemic injury; however, an understanding of the underlying HOPE mechanism that protects mitochondria is somewhat lacking. We hypothesized that mitophagy may play an important role in HOPE mitochondria protection. Experimental rat liver grafts were exposed to 30 min of in situ warm ischemia. Then, grafts were procured, followed by cold storage for 3 or 4 h to mimic the conventional preservation and transportation time in donation after circulatory death (DCD) in clinical contexts. Next, the grafts underwent hypothermic machine perfusion (HMP) or HOPE for 1 h through portal vein only perfusion. The HOPE-treated group showed a better preservation capacity compared with cold storage and HMP, preventing hepatocyte damage, nuclear injury, and cell death. HOPE can increase mitophagy marker expression, promote mitophagy flux via the PINK1/Parkin pathway to maintain mitochondrial function, and reduce oxygen free radical generation, while the inhibition of autophagy by 3-methyladenine and chloroquine could reverse the protective effect. HOPE-treated DCD liver also demonstrated more changes in the expression of genes responsible for bile metabolism, mitochondrial dynamics, cell survival, and oxidative stress. Overall, HOPE attenuates hypoxia-ischemic injury in DCD liver by promoting mitophagy flux to maintain mitochondrial function and protect hepatocytes. Mitophagy could pave the way for a protective approach against hypoxia-ischemic injury in DCD liver.

Intestinal Microbiota Participates in the Protective Effect of HO-1/BMMSCs on Liver Transplantation With Steatotic Liver Grafts in Rats.

The present study aimed to explore whether heme oxygenase-1 (HO-1)-modified bone marrow mesenchymal stem cells (BMMSCs) have a protective effect on liver transplantation with steatotic liver grafts in rats, and to determine the role of the intestinal microbiota in such protection. HO-1/BMMSCs were obtained by transduction of Hmox1 gene [encoding heme oxygenase (HO-1)]-encoding adenoviruses into primary rat BMMSCs. Steatotic livers were obtained by feeding rats a high-fat diet, and a model of liver transplantation with steatotic liver grafts was established. The recipients were treated with BMMSCs, HO-1/BMMSCs, or neither, via the portal vein. Two time points were used: postoperative day 1 (POD 1) and POD 7. The results showed that under the effect of HO-1/BMMSCs, the degree of steatosis in the liver grafts was significantly reduced, and the level of liver enzymes and the levels of pro-inflammatory cytokines in plasma were reduced. The effect of HO-1/BMMSCs was better than that of pure BMMSCs in the prolongation of the rats' postoperative time. In addition, HO-1/BMMSCs promoted the recovery of recipients' intestinal structure and function, especially on POD 7. The intestinal villi returned to normal, the expression of tight junction proteins was restored, and intestinal permeability was reduced on POD 7. The intestinal bacterial of the LT group showed significantly weakened energy metabolism and overgrowth. On POD 1, the abundance of Akkermansiaceae was higher. On POD 7, the abundance of Clostridiaceae increased, the level of lipopolysaccharide increased, the intestinal mucosal barrier function was destroyed, and the levels of several invasive bacteria increased. When treated with HO-1/BMMSCs, the energy metabolism of intestinal bacteria was enhanced, and on POD 1, levels bacteria that protect the intestinal mucosa, such as Desulfovibrionaceae, increased significantly. On POD 7, the changed intestinal microbiota improved lipid metabolism and increased the levels of butyrate-producing bacteria, such as Lachnospiraceae. In conclusion, HO-1/BMMSCs have protective effects on steatotic liver grafts and the intestinal barrier function of the recipients. By improving lipid metabolism and increasing the abundance of butyrate-producing bacteria, the changed intestinal microbiota has a protective effect and prolongs the recipients' survival time.

Exosome is involved in liver graft protection after remote ischemia reperfusion conditioning

BackgroundRemote ischemic perconditioning (RIPerC) has been demonstrated to protect grafts from hepatic ischemia-reperfusion injury (IRI). This study investigated the role of exosomes in RIPerC of liver grafts in rats. MethodsTwenty-five rats (including 10 donors) were randomly divided into five groups (n = 5 each group): five rats were used as sham-operated controls (Sham), ten rats were for orthotopic liver transplantation (OLT, 5 donors and 5 recipients) and ten rats were for OLT + RIPerC (5 donors and 5 recipients). Liver architecture and function were evaluated. ResultsCompared to the OLT group, the OLT + RIPerC group exhibited significantly improved liver graft histopathology and liver function (P < 0.05). Furthermore, the number of exosomes and the level of P-Akt were increased in the OLT + RIPerC group. ConclusionsRIPerC effectively improves graft architecture and function, and this protective effect may be related to the increased number of exosomes. The upregulation of P-Akt may be involved in underlying mechanisms.

Sirtuin-1 expression and activity is diminished in aged liver grafts

The cellular mechanisms underlying impaired function of aged liver grafts have not been fully elucidated, but mitochondrial dysfunction appears to be contributory. Sirtuin1 has been identified as a key mediator of mitochondrial recovery following ischemia–reperfusion injury. The purpose of this study was to determine whether differences exist in sirtuin-1 expression/activity in old vs. young liver grafts and to determine correlations with mitochondrial function, graft metabolic function, and graft injury. Old and young rat liver grafts (N = 7 per group) were exposed to 12 h of static cold storage (SCS), followed by a 2 h period of graft reperfusion ex vivo. Sirtuin1 expression and activity, mitochondrial function, graft metabolic function, and graft injury were compared. Sirtuin1 expression is upregulated in young, but not old, liver grafts in response to cold storage and reperfusion. This is associated with diminished tissue ATP, antioxidant defense, and graft metabolic function in old liver grafts. There was no evidence of increased inflammation or histologic injury in old grafts. Sirtuin1 expression is diminished in old liver grafts and correlates with mitochondrial and metabolic function. The sirtuin pathway may represent a target for intervention to enhance the function of aged liver grafts.

Repopulation of intrahepatic bile ducts in engineered rat liver grafts.

Engineered liver grafts for transplantation with sufficient hepatic function have been developed both in small and large animal models using the whole liver engineering approach. However, repopulation of the bile ducts in the whole liver scaffolds has not been addressed yet. In this study, we show the feasibility of repopulating the bile ducts in decellularized rat livers. Biliary epithelial cells were introduced into the bile ducts of the decellularized liver scaffolds with or without hepatocytes in the parenchymal space. The recellularized grafts were cultured under perfusion for up to 2 days and histological analysis revealed that the biliary epithelial cells formed duct-like structures, with the viable hepatocyte mass residing in the parenchymal space, in an arrangement highly comparable to the native tissue. The grafts were viable and functional as confirmed by both albumin and urea assay results and the gene expression analysis of biliary epithelial cells in recellularized liver grafts. This study provides the proof-of-concept results for rat liver grafts co-populated with parenchymal and biliary epithelial cells.

Improvement of Normothermic Ex Vivo Machine Perfusion of Rat Liver Grafts by Dialysis and Kupffer Cell Inhibition With Glycine

Normothermic ex vivo liver machine perfusion might be a superior preservation strategy for liver grafts from extended criteria donors. However, standardized small animal models are not available for basic research on machine perfusion of liver grafts. A laboratory-scaled perfusion system was developed consisting of a custom-made perfusion chamber, a pressure-controlled roller pump, and an oxygenator. Male Wistar rat livers were perfused via the portal vein for 6 hours using oxygenated culture medium supplemented with rat erythrocytes. A separate circuit was connected via a dialysis membrane to the main circuit for plasma volume expansion. Glycine was added to the flush solution, the perfusate, and the perfusion circuit. Portal pressure and transaminase release were stable over the perfusion period. Dialysis significantly decreased the potassium concentration of the perfusate and led to significantly higher bile and total urea production. Hematoxylin-eosin staining and immunostaining for single-stranded DNA and activated caspase 3 showed less sinusoidal dilatation and tissue damage in livers treated with dialysis and glycine. Although Kupffer cells were preserved, tumor necrosis factor α messenger RNA levels were significantly decreased by both treatments. For proof of concept, the optimized perfusion protocol was tested with donation after circulatory death (DCD) grafts, resulting in significantly lower transaminase release into the perfusate and preserved liver architecture compared with baseline perfusion. In conclusion, our laboratory-scaled normothermic portovenous ex vivo liver perfusion system enables rat liver preservation for 6 hours. Both dialysis and glycine treatment were shown to be synergistic for preservation of the integrity of normal and DCD liver grafts.

Optimized postconditioning algorithm protects liver graft after liver transplantation in rats

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

Fibrosis in small syngeneic rat liver grafts because of damaged bone marrow stem cells from chronic alcohol consumption.

A patient with liver failure due to chronic and acute alcohol abuse under consideration for an urgent liver transplant shortly after stopping alcohol may have residual abnormalities that threaten transplant success, particularly for a small graft. To address this, we studied a model in which reduced-size (50%) Lewis rat livers are transplanted into green fluorescence protein transgenic Lewis recipients after they are fed alcohol or a control diet for 5 weeks. Here we show that normal small Lewis grafts transplanted to alcohol-fed Lewis hosts developed fibrosis, whereas no fibrosis was observed in control-fed recipients. Host-derived CD133 + 8-hydroxy-2'-deoxyguanosine (8-OHdG) cells were significantly increased in livers recovered from both alcohol-fed and control recipients, but only alcohol-fed recipients demonstrated co-staining (a marker of oxidative DNA damage). α smooth muscle actin (α-SMA) staining, a marker for myofibroblasts, also co-localized with CD133 + cells only in the livers of alcohol-fed recipients. Immunostaining and polymerase chain reaction analysis confirmed that chronic alcohol consumption decreased the proportion of bone marrow stem cells (BMSCs) expressing CD133, c-Kit, and chemokine (C-X-C motif) receptor 4 markers and caused oxidative mitochondria DNA (mtDNA) damage. Culture of CD133 + cells from normal rats with medium containing 3% ethanol for 48 hours resulted in elevated mitochondrial 8-OHdG and mtDNA deletion, and ethanol exposure diminished CD133 expression but dramatically increased α-SMA expression. In conclusion, oxidative mtDNA damage and deletions occur in BMSCs of chronic alcohol-fed recipients, and these damaged cells mobilize to the small liver grafts and become myofibroblasts where they play a key role in the subsequent development of fibrosis. Liver Transplantation 23 1564-1576 2017 AASLD.

Hypothermic oxygenated perfusion (HOPE) for fatty liver grafts in rats and humans

Pretreatment of marginal organs by perfusion is a promising opportunity to make more organs available for transplantation. Protection of human donation after cardiac death (DCD) livers by a novel machine perfusion technique, hypothermic oxygenated perfusion (HOPE), was recently established. Herein, we tested whether HOPE is also useful for fatty liver grafts, using a rodent transplant model. Rats were fed over three weeks with a special methionine-choline-deficient diet (MCDD) to induce severe hepatic macrosteatosis (≥60%). Afterwards, livers were transplanted with either minimal or 12h cold storage. Additional liver grafts were treated after 12h cold storage with 1h HOPE before transplantation. Graft injury after orthotopic liver transplantation (OLT) was assessed in terms of oxidative stress, damage-associated molecular patterns release, toll-like receptor-4 activation, cytokine release, endothelial activation, and the development of necrosis and fibrosis. Implantation of cold stored macrosteatotic liver grafts induced massive reperfusion injury after OLT, compared to controls (non-fatty livers). HOPE treatment after cold storage failed to change the degree of steatosis itself, but markedly decreased reperfusion injury after OLT, as detected by less oxidative stress, less nuclear injury, less Kupffer- and endothelial cell activation, as well as less fibrosis within one week after OLT. Protective effects were lost in the absence of oxygen in the HOPE perfusate. HOPE after cold storage of fatty livers prevents significant reperfusion injury and improves graft function, comparable to the effects of HOPE in DCD livers and DCD kidneys. HOPE treatment is easy and may become a universal concept to further expand the donor pool. An increasing number of donor livers contain fat. It is important to harness marginal livers, which may contain fat, as the stock of donor livers is limited. Hypothermic oxygenated perfusion (HOPE) prevents reperfusion injury and improves liver graft function. HOPE offers a simple and low-cost option for treating liver grafts in transplant centers, even after cold storage, instead of transporting machines to the place of procurement. HOPE could be used globally to expand the donor pool.

Nitrite enhances liver graft protection against cold ischemia reperfusion injury through a NOS independent pathway

ABSTRACTIntroduction: Nitrite has been found to protect liver graft from cold preservation injury. However, the cell signaling pathway involved in this protection remains unclear. Here, we attempt to clarify if the NOS pathway by using the NOS inhibitor, L-NAME (L-NG-Nitroarginine methyl ester).Animals and methods: Rat livers were conserved for 24 h at 4°C in (IGL-1) solution enriched or not with nitrite at 50 nM. In a third group, rats were pretreated with 50 mg/kg of L-NAME before their liver procurement and preservation in IGL-1 supplemented with nitrite (50 nM) and L-NAME (1 mM). After 24 h of cold storage, rat livers were ex-vivo perfused at 37°C during 2 h. Control livers were perfused without cold storage.Results: Nitrite effectively protected the rat liver grafts from the onset of cold I/R injury. L-NAME treatment did not abolish the beneficial effects of nitrite. Liver damage, protein oxidation and lipid peroxidation remained at low levels in both nitrite-treated groups when compared to IGL-1 group. Antioxidant enzyme activities and functional parameters were unchanged after NOS inhibition.Conclusion: Despite NOS inhibition by L-NAME, nitrite can still provide hepatic protection during cold I/R preservation. This suggests that nitrite acts through a NOS-independent pathway.

Impact of Splenectomy Just Before Partial Orthotopic Liver Transplantation Using Small-for-Size Graft in Rats

BackgroundPortal hypertension is a serious obstacle of partial orthotopic liver transplantation (POLT) with the use of small-for-size liver graft. Several therapeutic strategies including surgical innovations and pharmacological agents to reduce the portal hypertension have been developed. Splenectomy (SP) on POLT is one of surgical procedures to reduce portal pressure. We previously reported a dual cytoprotective mechanism of SP just before POLT, using small-for-size liver graft in a rat model. However, the best timing of SP during POLT has been unclear. We compared liver functions between SP just before and after POLT, using small-for-size rat liver grafts. MethodsWith the use of small-for-size liver grafts (20%) in rats previously reported, the rats were assigned to 2 groups: the pre-SP group (SP just before POLT) and the post-SP group (SP just after POLT). Liver tissues and blood were sampled at 6 and 24 hours after POLT for several liver function tests. ResultsThe serum alanine aminotransferase levels at 24 hours after POLT were significantly decreased in the pre-SP group compared with the post-SP group (226 ± 78 vs 340 ± 71 IU/L). The infiltrations of neutrophil at 6 hours and ED-1–positive cells at 24 hours were significantly suppressed in the pre-SP group compared with the post-SP group. Serum hyaluronic acid levels, indicating attenuation of endothelial damage, were lower in the pre-SP group than in the post-SP group. ConclusionsSP before POLT, which directly eliminates splenic inflammatory leukocytes, inhibits inflammatory leukocyte infiltration, which leads to impaired liver function as compared with SP after POLT.

Protective effects of hydrogen-enriched saline against ischemia-reperfusion injury in non-heart-beating rat liver graft

Journal of the American College of Surgeons 221(4):p e102-e103, October 2015. | DOI: 10.1016/j.jamcollsurg.2015.08.171

Innovative Pharmacological/Therapeutic Approaches against Hepatic Ischemia/Reperfusion Injury.

Prolonged oxygen deprivation corresponding to the ischemic period is observed during liver resection, transplantation, and trauma and subsequent oxygen restoration always leads to reperfusion injury. In particular, the ischemic period represents an inevitable event during conventional organ preservation before transplantation and the magnitude and severity of reperfusion injury depend on the time of liver preservation. Faced with the increasing shortage of donor organs for transplantation, there is renewed interest in marginal livers, such as those obtained from donation after circulatory death or fatty livers, which are particularly susceptible to ischemia/reperfusion (I/R) injury. The decreased tolerance towards I/R observed in fatty livers has not been fully elucidated yet, and underlying mechanisms appear different in lean and steatotic livers. The paper of A. Matsuda et al. proposed the use of activated protein C (APC), an anticoagulant, found to induce an initial attenuation of tissue damage by inhibiting inflammatory cell infiltration and sinusoidal endothelial injury in normal liver. Of interest, in steatotic mice livers, APC did not affect initial liver damage but attenuated late damage, suggesting the existence of an additional pathway to the anti-inflammatory cytoprotective effect of APC which may occur via activation of adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. A further attempt to reduce the fatty liver susceptibility was reported by E. Pantazi et al. that used a peroxisome proliferator-activated receptor α (PPARα) agonist WY-14643. PPARα is a nuclear receptor highly expressed in liver and it functions as a lipid sensor. Treatment with WY-14643 reduced liver injury in fatty rat livers, enhanced the deacetylase enzyme sirtuin 1 (SIRT1), recently found to be a target for preventing I/R, and prevented endoplasmic reticulum stress. M. Bejaoui et al. investigated the use of polyethylene glycols (PEGs) that, besides their usefulness as oncotic agents in preservation solution, have been shown to protect against cold injury and ischemic damage. The intravenous PEG 35 administration by a unique dose protected steatotic rat liver grafts against the deleterious effects of cold storage and the subsequent reperfusion. The rationale for the intravenous administration of PEG 35 was to induce a pharmacological preconditioning effect; its protective mechanisms are related to preservation of mitochondria and the induction of protective cell signaling pathways (eNOS, Akt, and AMPK). An additional model of liver preconditioning by pharmacological induction of adenosine A2a receptor (A2aR) was reported by E. Alchera et al. The analysis of the molecular changes induced by A2aR stimulation revealed multiple mechanisms of liver cell protection that can be both immediate (early preconditioning) and delayed (late preconditioning). Note that the A2aR activation also protects against lipotoxicity by preventing lipoapoptosis in primary rat hepatocytes. Among the hepatic mechanisms involved in the development of I/R, mitochondrial dysfunction represents a crucial cellular event contributing to I/R injury. As reported by K. Go et al., the elimination of abnormal and dysfunction mitochondria by mitophagy appears to increase both the quality of the mitochondria and cell survival. Although the mechanisms underlying the onset and propagation of mitophagy remain elusive, this event represents an early adaptive response to facilitate cell survival during I/R. Articles published in this issue not only help to identify new pharmacological approaches for improving liver function during ischemia/reperfusion damage but also stimulate the identification of molecular mediators of hepatic injury. Mariapia Vairetti Hartmut Jaeschke Diethard Monbaliu Jae-Sung Kim Andrea Ferrigno

Normothermic Perfusion Preservation Protects Non-Parenchymal Cells of Liver Grafts in Rats.

Normothermic Perfusion Preservation Protects Non-Parenchymal Cells of Liver Grafts in Rats.

Normothermic Acellular Machine Perfusion and Bile Duct Injury in Pig Livers Retrieved After Cardiac Death

To the Editor: We have read with interest the article by Boehnert et al (1Boehnert MU Yeung JC Bazerbachi F et al.Normothermic acellular ex vivo liver perfusion reduces liver and bile duct injury of pig livers retrieved after cardiac death.Am J Transplant. 2013; 13: 1441-1449Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), describing normothermic acellular machine perfusion of pig livers donated after cardiac death (DCD). The authors should be commended for performing this important study which adds to the evidence that normothermic machine perfusion (NMP) is a promising method to reduce biliary injury after transplantation of DCD livers. Using an erythrocyte-based perfusion fluid, our group previously reported the feasibility of NMP of human DCD livers, emphasizing that this preservation method is about to enter the clinical arena (2Op den Dries S Karimian N Sutton ME et al.Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.Am J Transplant. 2013; 13: 1327-1335Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Nevertheless, there are still some unanswered questions that are not fully addressed by Boehnert et al (1Boehnert MU Yeung JC Bazerbachi F et al.Normothermic acellular ex vivo liver perfusion reduces liver and bile duct injury of pig livers retrieved after cardiac death.Am J Transplant. 2013; 13: 1441-1449Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar).–In contrast to previous studies on NMP, Boehnert et al did not use erythrocytes as oxygen carrier during NMP. The amount of oxygen that can be dissolved in aqueous fluids at 38°C is very low and it can be questioned whether sufficient amounts of oxygen are provided to the liver using an acellular perfusion fluid at 38°C. The authors present a method for calculating oxygen extraction that uses Henry’s constant for O2 in water at 25°C and does not correct for liver mass. Contrary to the described method, oxygen consumption is inappropriately expressed in mmHg (Results section and Figure 4) instead of a unit that takes flow and liver mass into account such as mL O2/g liver/min (3Tolboom H Pouw RE Izamis ML et al.Recovery of warm ischemic rat liver grafts by normothermic extracorporeal perfusion.Transplantation. 2009; 87: 170-177Crossref PubMed Scopus (71) Google Scholar). Because of this discrepancy between method and results, we question whether calculations of oxygen extraction were performed correctly.–Another aspect that raises questions is the preservation of bile ducts. Boehnert et al reported 100% necrosis of bile duct mucosa after reperfusion of DCD livers subjected to 60 min of donor warm ischemia and 4 h of static cold storage (CS). However, only one out of five DCD livers subjected to 60 min donor warm ischemia and 4 h CS plus 8 h NMP was reported to have minor bile duct injury after blood reperfusion. The other four bile ducts in this group demonstrated 100% intact mucosal lining without any necrosis. First, it remains unclear why biopsies of only five bile ducts were analyzed, whereas six livers were included in each group. Second, the results suggest that extension of the preservation time by 8 h of NMP, using oxygenated Steen solution, can almost completely protect bile ducts against universal cell death due to 60 min warm ischemia plus 4 h CS, followed by blood reperfusion. Such an impressive difference is difficult to understand.–Although bile flow has been indicated as the most important parameter of liver viability during NMP (4Imber CJ St Peter SD Lopez de Cenarruzabeitia I et al.Advantages of normothermic perfusion over cold storage in liver preservation.Transplantation. 2002; 73: 701-709Crossref PubMed Scopus (198) Google Scholar), bile production after either ex vivo reperfusion or transplantation was very low (1–2 mL/h) and no significant differences in bile flow were noted between NMP and CS. This raises the question how effective hepatic function was preserved by the presented NMP system.–Finally, it remains unclear why only bile flow and serum AST, and not bile duct histology or any other marker of function or injury, were presented as outcome parameters in the transplant model. The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Chronic Antibody-Mediated Responses May Mediate Chronic Rejection in Rat Orthotopic Liver Transplantation

The category of chronic antibody-mediated rejection (AMR) is not included in Banff schema for liver allograft rejection. In the present study, we examined the pathology of chronic rejection using rat liver transplantation. Orthotopic liver transplantation from Lewis to BN rats was performed without immunosuppression, and with or without HA reconstruction. We studied grafts at day 120 for arterialized and day 39 for nonarterialized transplants focusing on the immunoglobulin G (IgG) deposition and the pathologic characteristics of rejection. About 20% of arterialized grafts survived more than 120 days. Between day 7 and day 120, T-cell infiltration to arterialized grafts was accompanied by IgG deposition in portal veins, hepatic arteries, and bile ducts in portal areas, sinusoids and hepatocytes. At day 120, arterialized grafts were morphologically characterized by late chronic rejection with IgG deposition, intraluminal portal veins fibrosis, intimal fibrous thickening of hepatic arteries, diffuse sinusoidal fibrosis, as well as injury and loss of bile ducts due to fibrosis. The severities of T cell-mediated rejection and AMR were higher in nonarterialized than arterialized grafts. Nonarterialized Lewis liver grafts in BN rats were rejected by day 39, as characterized by late chronic rejection with IgG deposition and cellular infiltration. In conclusion, chronic AMR may be involved in chronic rejection of liver transplantations. When chronic AMR was involved in chronic liver graft rejection, typical late morphological changes emerged within a short period.

Changes in peripheral blood Th1 and Th2 cells in rat liver transplantation under different immune statuses

In this study, early expressions of peripheral blood Th1 and Th2 cells were documented following rat liver transplantation and related to immune status. Rats were divided into 3 groups: group A (control): syngeneic transplantation (Brown Norway (BN) → BN); group B: allogeneic transplantation + cyclosporine A (CsA); group C: allogeneic transplantation (Lewis → BN). Flow cytometry was used to analyze peripheral blood CD4(+)CD45RC percentage on days 1, 3, 5, 7, and 14 following transplantation, and were compared to graft rejection pathological grades and receptor survival times. The average survival of groups A and B exceeded 100 days, which was significantly longer than that of group C (3.56 ± 34.3 days). With the exception of the first day, rejection grades were significantly higher in groups C and B compared to group A, and group C rejection grades were significantly higher than those of group B. Three days after transplantation, the CD4(+)CD45RC(+) to CD4(+)CD45RC(-) ratio of group C was significantly higher than that of groups A and B. In group B, the CD4(+)CD45RC(+) to CD4(+)CD45RC(-) ratio was negatively correlated to the rejection grade (r = -0.565, P < 0.01), whereas this relationship was positive in group C (r = 0.745, P < 0.01). In conclusion, peripheral blood Th1 was highly expressed during rejection in rat liver grafts. Peripheral blood Th2 tended to increase early under rejection inhibition with CsA, and its high expression level may correlate with long-term acceptance or tolerance of transplanted livers.