Graft calcifications and dysfunction following liver transplantation

Background  

The molecular events, following ischemia and reperfusion (I/R) of the liver during transplantation are largely unknown. There is evidence that apoptotic and necrotic events may take place, and occasionally result in primary graft dysfunction. We herein report two cases, where significant I/R injury correlated with the development of liver calcification and primary liver dysfunction.     

Role of Ischemic Preconditioning in Liver Surgery and Hepatic Transplantation

Introduction  

The purpose of this review is to summarize intraoperative surgical strategies available to decrease ischemia–reperfusion injury associated with liver resection and liver transplantation.     

Evaluation of microperfusion disturbances in the transplanted liver after Kupffer cell destruction using GdCl3: an experimental porcine study

Background

Organ function after liver transplantation is determined by ischemia-reperfusion injury. Destruction of Kupffer cells with gadolinium chloride (GdCl₃) has been shown to have a possible preventive effect on the extent of this injury, which can be extrapolated by analyzing the distribution of hepatic microperfusion. The aim of this study was to evaluate the protective effect of GdCl₃ on disturbances of microperfusion in the transplanted liver.

Methods

Landrace pigs were randomly divided into three groups. In the control group (CG; n = 6) a mapping of the native liver was conducted. For mapping, the four hepatic liver lobes were named from right to left with A to D and every lobe was divided into three vertical segments (cranial, medial, and caudal). In each of these 12 areas, microperfusion was quantified using a thermodiffusion probe (TD [mL/100 g/min]). The other two groups were considered as transplanted treated group (TTG; n = 10) and transplanted nontreated group (TnTG; n = 10). The TTG received an infusion of 20 mg/kg GdCl₃ intravenously 24 hours before organ harvesting. Then standardized orthotopic liver transplantation was performed. In TnTG, standardized orthotopic liver transplantation was carried out without prior GdCl₃ injection. In the recipients, the microperfusion of transplanted livers were mapped in both TnTG and TTG, in two different time points (1 hour [n = 5] and 24 hours (n = 5]) after reperfusion.

Results

A significant reduction of macrophages in the TTG livers in comparison to the CG and TnTG livers was observed (P < .05). However, the number of macrophages in CG and TnTG livers showed no significant difference (P > .05). Regarding liver microperfusion, in TnTG, a marked heterogeneity was detected in the livers after reperfusion. Significant differences between liver lobes (horizontal planes; P = .032) and vertical layers of intralobar liver parenchyma (P = .029) were observed. The same pattern was seen in TTG livers after reperfusion and a significant difference between horizontal (P = .024) and vertical layers (P = .018) of liver tissue were observed. Comparing intralobar regional flow data between vertical planes 24 hours after reperfusion still showed a prominent variation of hepatic tissue perfusion in TnTG livers (P = .028). Within the same horizontal layers, no significant differences between lobes were measured anymore (P = .16). Contrary to TnTG, in TTG, a homogenous pattern of regional liver tissue perfusion was recorded 24 hours after reperfusion. Comparison of TD data on the liver regions showed no significant microperfusion differences in either horizontal (P = .888) or vertical (P = .841) layers.

Conclusions

Application of GdCl₃ resulted in a significant reduction of Kupffer cells. Twenty four hours after transplantation microperfusion showed a homogeneous pattern, which constituted an earlier and better recovery of the transplanted liver. Therefore, destruction of Kupffer cells reduced ischemia-reperfusion injury and seemed to be responsible for the early recovery of microperfusion disturbances and thus for an improvement of graft function.     

Relationship Between Ischemia/Reperfusion Injury and Acute Rejection of Allogeneic Liver Transplant in Rats

Objective

This study aimed to investigate the relationship between the severity of ischemia/reperfusion (I/R) injury and the acute rejection (AR) of allogeneic liver transplants in rats.

Methods

The experimental rats were divided in different groups: normal control group (sham group, group I); syngeneic liver transplant control group (similar gene group, group II); and allogeneic liver transplant groups (groups III to VI). The rats were humanely killed at 1, 3, 5, and 7 days after transplantation or sham operation to determine the severity of I/R injury, rejection classification, and hepatocyte apoptosis. Messenger RNA (mRNA) and protein expression levels of Fas, perforin, and granzyme B were assessed in the liver tissues using real-time polymerase chain reaction and immunohistochemistry, respectively.

Results

The rejection scores of the transplanted liver tissues gradually increased until these scores were proportional to the severity of I/R injury in groups III, IV, and V. The maximum scores were reached at 7 days after transplantation as the duration of transplantation was extended. The mRNA and protein expression levels of Fas, perforin, and granzyme B were significantly increased at 1, 3, 3, 5, and 7 days after liver reperfusion in groups III, IV, and V compared with those in groups I, II, and VI (P < .05).

Conclusion

The occurrence of AR after allogeneic liver transplantation in rats was positively correlated with the severity of I/R injury. Given that I/R injury caused serious damage to the transplanted liver, the occurrence of AR consequently decreased.     

Taurine improves graft survival after experimental liver transplantation.

Relevant mechanisms of reperfusion injury after liver transplantation are most likely mediated by activated Kupffer cells. Recently, it has been demonstrated that taurine prevents Kupffer cell-activation in vitro. Thus, this study was designed to assess the effects of taurine after liver transplantation. Female Sprague-Dawley rats (210-240 g) were infused with taurine dissolved in normal saline, before organ harvest. Controls were infused with the same volume of normal saline without taurine. Following 4 hours of cold ischemia, liver transplantation was performed. Graft and animal survival, serum transaminases, liver histology, perfusion data of intravital microscopy, blood distribution at reperfusion, and both phagocytosis of Kupffer cells and expression of tumor necrosis factor alpha (TNF-alpha) to index cellular activation were investigated. For comparison, both, analysis of variance (ANOVA) and Fisher's exact test were used as appropriate. Results are presented as mean +/- SEM. Controls survived in 60% of cases. Taurine improved survival in a dose-dependent manner to 100% (P < 0.05). In controls, mean aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) serum levels increased to 3,260 +/- 814; 1,703 +/- 432; and 14,071 +/- 3,177 U/L, respectively, after transplantation. In contrast, these values were between 20 and 45% of control values after taurine (P < 0.05). Histology taken after transplantation confirmed the significant protective effects of taurine, including the reduction of TNF-alpha expression. Time until homogeneous reperfusion of the graft improved to 50% of control values (P < 0.05). Further, taurine significantly decreased both phagocytosis of latex beads by Kupffer cells and leukocyte-endothelial cell interaction. In parallel, flow velocity of red blood cells as well as acinar and sinusoidal perfusion improved (P < 0.05). In conclusion, these data show for the first time in vivo that taurine minimizes reperfusion injury after liver transplantation. Decreased leukocyte-endothelial cell interaction and improved microcirculation are the proposed mechanisms, which are most likely Kupffer cell-dependent.     

Donor pretreatment with gadolinium chloride improves early graft function and survival after porcine liver transplantation

Kupffer cell depletion by gadolinium chloride (GdCl₃) in rat livers has previously been proven to minimize hepatic ischemia/reperfusion injury after experimental liver transplantation (LTX). In the current study, we evaluated the effects of donor pretreatment with GdCl₃ on hepatic ischemia/reperfusion injury, macro- and microcirculation, and endotoxin clearance of the liver in a porcine model of experimental LTX. Two groups of 12 pigs were treated either with intravenous NaCl (0.9%; control) or GdCl₃ (20 mg/kg). Twenty-four hours after pretreatment, hepatic macrocirculation was quantified by Doppler flowmetry and liver parenchymous microcirculation by implanted thermodiffusion electrodes. The liver grafts were transplanted after 4–6 h of cold ischemia in University of Wisconsin (UW) solution. At 1 and 24 h after LTX, the perfusion values were re-evaluated and histology, biochemical (aspartate aminotransferase, AST) and functional parameters (partial thromboplastin time, prothrombin time, and bilirubin) were analyzed. Furthermore, endotoxin clearance of the liver was evaluated at all time points. In GdCl₃-treated animals 80% of the Kupffer cells were destroyed, and 24 h after LTX ischemia/reperfusion injury in treated grafts was significantly lower in comparison to controls, as shown by histology, AST levels (741±490 U/l in controls vs 379±159 U/l in treated grafts, P<0.05), survival (67% vs 92%), and enhanced macro- (total transhepatic blood flow [THBF]=112±22 ml/min per 100 g in controls vs 157±45 ml/min per 100 g in treated grafts, P0.05) and microcirculation (thermodiffusion [TD]=73±9 ml/min per 100 g in controls vs 90±16 ml/min per 100 g in treated grafts, P0.05). Despite destruction of the macrophage system in the liver, the transhepatic endotoxin gradient of treated livers was enhanced before and 1 h after transplantation (58% in controls vs 85% in treated grafts, P<0.05). Destruction of Kupffer cells of donors by pretreatment with GdCl₃ in pigs is effective in preventing liver graft dys- and nonfunction after LTX. Pretreatment with GdCl₃ does not diminish but increase hepatic endotoxin clearance.     

HTK-N, a modified HTK solution, decreases preservation injury in a model of microsteatotic rat liver transplantation

Background

Ischemia/reperfusion injury is an obstacle especially in steatotic livers, including those with steatosis induced by acute toxic stress. Recently, a modified histidine-tryptophan-ketoglutarate (HTK) solution, HTK-N, has been developed. This solution contains N-acetylhistidine, amino acids, and iron chelators. This study was designed to test the effects of HTK-N on preservation injury to rat livers after acute toxic injury.

Methods

Microvesicular steatosis was induced by a single dose of ethanol (8?g/kg BW). Livers were harvested and stored at 4?°C for 8?h with HTK or HTK-N before transplantation. Tissue and blood samples were taken at 1, 8, and 24?h after reperfusion to compare serum liver enzymes (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase), standard histology, and immunohistochemistry for myeloperoxidase (MPO), caspase-3, and inducible nitric oxide synthase. Survival was compared after 1?week. For statistics, Analysis of Variance and t test were used.

Results

HTK-N improved survival from 12.5?% in HTK to 87.5?% (p?<?0.05). Furthermore, liver enzymes were decreased to 2?C75?% of HTK values (p?<?0.05). Necrosis and leukocyte infiltration and MPO, caspase-3, and iNOS expression after transplantation were decreased (p?<?0.05).

Conclusions

This study demonstrates that HTK-N protects liver grafts with microvesicular steatosis caused by acute toxic injury from cold ischemic injury better than standard HTK most likely via inhibition of hypoxic injury and oxidative stress and amelioration of the inflammatory reaction occurring upon reperfusion.     

Early innate immune events induced by prolonged cold ischemia exacerbate allograft vasculopathy

Background  

Ischemia/reperfusion induced innate immune injury is inescapable in solid organ transplantation. Prolonged cold ischemia exacerbates the primary manifestation of late graft rejection, allograft vasculopathy (AV). The relationship between prolonged cold ischemia and late graft events is unclear and the subject of this study.     

Effect of taurine on IRAK4 and NF-kappa B in Kupffer cells from rat liver grafts after ischemia-reperfusion injury

BackgroundThe aim of this study was to explore the protective mechanisms of taurine pretreatment against hepatic ischemia/reperfusion injury after liver transplantation.MethodsA Sprague-Dawley–to–Sprague-Dawley rat liver transplantation model was used in this study. At 0, 60, and 180 minutes after reperfusion, expression of interleukin-1 receptor–associated kinase-4 (IRAK-4) messenger ribonucleic acid and protein in Kupffer cells was determined by real-time polymerase chain reaction and Western blotting. The activity of nuclear factor κB in Kupffer cells was determined by electrophoretic mobility shift assay. The serum tumor necrosis factor–α level was detected by enzyme-linked immunosorbent assay. Serum transaminases, liver histology, and animal survival were also investigated.ResultsAt 60 and 180 minutes after reperfusion, levels of IRAK-4 messenger ribonucleic acid and protein, activities of nuclear factor κB, and levels of serum transaminases and tumor necrosis factor–α were all obviously elevated. However, changes in these parameters in rats treated with taurine were remarkably attenuated at the indicated time points.ConclusionsThese data suggest that taurine could protect against hepatic ischemia/reperfusion injury after liver transplantation, and the protective effects may be through downregulation of IRAK-4 and downstream nuclear factor κB and tumor necrosis factor–α expression in Kupffer cells.     

Ischemic versus pharmacologic hepatic preconditioning

Background

Hepatic ischemia–reperfusion injury has a significant impact on liver resection and transplantation. Many strategies have been developed to reduce the effects of ischemia–reperfusion injury, including pharmacologic and ischemic preconditioning; however, studies comparing these two methods are lacking.

Material and methods

An experimental study was performed in a swine model. Eighteen swine were randomly assigned to three different groups: an ischemic preconditioning (IschPC) group, a pharmacologic preconditioning (PharmPC) group, and a control group. All animals underwent a 40-min liver ischemia, followed by 40 min of reperfusion. The IschPC group received a short period of ischemia (10 min) and a short period of reperfusion (15 min) before prolonged ischemia. The PharmPC group received inhaled sevoflurane for 30 min before prolonged ischemia. The control group did not receive any intervention before prolonged ischemia. Blood samples and liver tissue were obtained after ischemic and reperfusion periods. Injury was evaluated by measure of DNA damage (using COMET assay) and serum biochemical markers (transaminases, alkaline phosphatase, amylase, bilirubin, and C-reactive protein [CRP]).

Results

No significant difference was found in serum biochemical markers, except for the C-reactive protein level that was lower in the PharmPC group than in the control group soon after hepatic ischemia. Soon after prolonged ischemia, DNA damage index, both in blood samples and in liver tissue samples, was similar among the groups. However, an increase in DNA damage after reperfusion was higher in the control group than in the PharmPC group (P < 0.05). The increase in DNA damage in the IschPC group was half of that observed in the control, but this difference was not statistically significant.

Conclusions

Our results suggest an early protective effect of PharmPC (lower levels of C-reactive protein soon after ischemia). The protective effect observed after reperfusion was higher with PharmPC than with ischemic preconditioning. The simultaneous use of both methods could potentiate protection for ischemia–reperfusion.     

A novel in situ model of liver cold ischemia–reperfusion in rats

Background

Orthotopic liver transplantation (OLT) is being used for studying cold ischemia reperfusion (I/R)-induced injury in experimental animals, but the technique is complicated and it does not accurately reflect the pathophysiology. Here, we report a novel model, termed “in situ liver cold ischemia (ISLCI)”, in Wistar rats.

Methods

ISLCI was achieved in rats by establishing a portal-jugular shunt and a cannula shunt in inferior vena cava, and the liver was continuously perfused with lactate Ringer's solution at a speed of 150 mL/h through the portal vein for 60 min. Portal venous pressure, serum levels of total bilirubin, alkaline phosphatase, alanine aminotransferase and γ-glutamyl transpeptidase (GGT), and hepatic histopathology were examined, and compared with rats undergoing OLT, in which the donor liver was subjected to a 60 min cold ischemia.

Results

Livers from ISLCI and OLT rats showed histopathologic changes characteristic of I/R-induced injury when examined on days 1 and 7, with complete recovery 14 d after reperfusion. Compared with OLT rats, ISLCI rats had significantly lower levels of portal venous pressure 1 and 10 min after porta hepatis clamping. They suffered a milder degree of I/R-induced hepatic injury, reflected by significantly lower levels of GGT, alanine aminotransferase, and alkaline phosphatase on day 1, and a significant lower level of GGT and a lower histopathologic score on day 7 after reperfusion.

Conclusions

Our preliminary results indicate that the ISLCI model is reliable and technically easier, and is superior to OLT for studying cold I/R injury.     

CXC chemokines play a critical role in liver injury, recovery, and regeneration

Background

Hepatic ischemia/reperfusion (I/R) injury is a principal consideration of trauma, resectional liver surgery, and transplantation. Despite improvements in supportive care, hepatic I/R injury continues to negatively impact patient outcomes because of significant tissue damage and organ dysfunction. CXC chemokines have been implicated as key mediators in the deleterious inflammatory cascade after hepatic I/R and also as important, beneficial regulators of liver recovery and regeneration. As such, their potential to mediate both beneficial and detrimental effects on hepatocytes makes them a key target for therapy. Herein, we provide a review of the inflammatory mechanisms of hepatic I/R injury, with a focus on the divergent functions of CXC chemokines in this response compared with other liver insults, and offer an explanation of this apparent paradox.

Data sources

MEDLINE and PubMed.

Conclusions

CXC chemokines are key mediators of both the inflammatory response to hepatic I/R as well as the recovery from this injury. Their contrasting functions in the regeneration of liver mass after an ischemic insult indicates that therapeutic manipulation of these mediator pathways should differ depending on the surgical milieu.     

阻断肝脏神经和抑制库普弗细胞对移植鼠肝保护作用的观察

目的　研究移植肝库普弗细胞和交感神经在再灌注损伤发生中的作用 ,以及阻断供肝交感神经和抑制库普弗细胞对移植肝的保护机制。方法　 13 0对实验大鼠随机分为 4组 ,即正常对照组 (N组 )、氯化钆组 (C组 )、六甲胺组 (H组 )和六甲胺氯化钆组 (HC组 )。同等条件下 ,于肝移植术后 4,8,16,2 4h检测受体的肝功能、肝储备功能 ,并切取肝脏组织行光镜、电镜检查。结果　阻断供肝交感神经和抑制供肝库普弗细胞可明显改善移植肝的肝功能和肝储备功能 ,形态学变化也与之相符。结论　阻断供肝交感神经和抑制供肝库普弗细胞对移植肝有保护作用     

Multidrug donor preconditioning prevents cold liver preservation and reperfusion injury

Purpose  

Primary graft dysfunction still represents a major challenge in liver transplantation. We herein studied in an isolated rat liver perfusion model whether a multidrug donor preconditioning (MDDP) can not only reduce but also completely prevent cold ischemia–reperfusion injury.     

Impact of oxygen free radicals in rat partial liver transplantation

Background

Due to the shortage of suitable organs, the demand for partial liver transplantation from living donors has increased worldwide. N-acetylcysteine (NAC) has shown protective effects as a free radical scavenger during hypothermic preservation and warm ischemia–reperfusion liver injury; however, no study has reported the effects in partial liver transplantation. The aim of this study was to analyze the impact of NAC on liver graft microcirculation and graft function after partial liver transplantation in rats.

Methods

Orthotopic partial liver transplantations were performed in 40 rats following cold storage in histidine-tryptophan-ketoglutarate solution for 3 h with 20 mM NAC (NAC group, n = 20) or without (control group, n = 20). We assessed portal circulation, graft microcirculation, and biochemical analyses of plasma at 1, 3, 24, and 168 h after portal reperfusion.

Results

(Control versus NAC, median and range): Portal venous pressure was significantly lower with NAC (P = 0.03). Microcirculation measured by laser Doppler was significantly improved with NAC throughout the time course (P = 0.003). Alanine aminotransferase levels were significantly lower in the NAC group (P < 0.05). Total antioxidative capacity was significantly higher in the NAC group at 1 h after reperfusion (Trolox equivalents: median, 3 μM; range, 2.9–6.7 versus median, 16.45 μM; range, 10.4–18.8). Lipid peroxidation was significantly abrogated in the NAC group (median, 177.6 nmol/mL; range, 75.9–398.1 versus median, 71.5 nmol/mL; range, 58.5–79 at 3 h).

Conclusions

This study showed that NAC treatment during cold storage resulted in improved microcirculation and preservation quality of partial liver graft likely because of enhanced antioxidant capacity and reduced lipid peroxidation.     

Perioperative infliximab application has marginal effects on ischemia–reperfusion injury in experimental small bowel transplantation in rats

Purpose  

Ischemia-reperfusion injury leads to impaired smooth muscle function and inflammatory reactions after intestinal transplantation. In previous studies, infliximab has been shown to effectively protect allogenic intestinal grafts in the early phase after transplantation with resulting improved contractility. This study was designed to reveal protective effects of infliximab on ischemia–reperfusion injury in isogenic transplantation.     

Large-for-size liver transplantation: a flowmetry study in pigs

Background

Ischemia–reperfusion injury is partly responsible for morbidity in pediatric liver transplantation. Large-for-size (LFS) liver transplantation has not been fully studied in the pediatric population, and the effects of reperfusion injury may be underestimated.

Materials and methods

Thirteen Landrace–Large white pigs weighing 23 kg (range, 17–38 kg) underwent orthotopic liver transplantation. They were divided into two groups according to the size of the donor body: LFS and control (CTRL). After transplantation, the abdominal cavity of the recipient was kept open and portal venous flow (PVF) was measured after 1 h. The ratio of recipient PVF (PVFr) to donor PVF was used to establish correlations with ischemia and reperfusion parameters. Liver biopsies were taken 1 h after transplantation to assess ischemia and reperfusion and to quantify the gene expression of endothelial nitric oxide synthase, interleukin 6, BAX, and BCL.

Results

Recipient weight, total ischemia time, and warm ischemia time were similar between groups. Among hemodynamic and metabolic analyses, pH, central arteriovenous PCO₂ difference, and AST were statistically worse in the LFS group than in the CTRL group. The same was found with endothelial nitric oxide synthase (0.41 ± 0.18 versus 1.56 ± 0.78; P = 0.02) and interleukin 6 (4.66 ± 4.61 versus 16.21 ± 8.25; P = 0.02). In the LFS group, a significant decay in the PVFr was observed in comparison with the CTRL group (0.93 ± 0.08 and 0.52 ± 0.11, respectively; P < 0.001).

Conclusions

The implantation of a graft was responsible for poor hemodynamic status of the recipient 1 h after transplantation. Furthermore, the LFS group demonstrated markers of ischemia and reperfusion that were worse when compared with the CTRL group and exhibited a more significant decrease in PVF from donor to recipient.     

枯否细胞在肝移植手术后缺血再灌注损伤中的作用

目的总结枯否细胞在肝移植术后缺血再灌注损伤中的作用。方法通过复习文献的方法对枯否细胞在缺血再灌注损伤中的作用进行综述。结果枯否细胞是肝内固有的巨噬细胞,肝移植手术后枯否细胞被激活释放一系列炎症介质,包括细胞因子、活性氧中间产物、趋化因子等,启动缺血再灌注损伤(ischemia reperfusioninjury,IRI),使移植肝失功。同时,枯否细胞不仅可以通过释放NO来减轻缺血再灌注损伤,也可以特异性的产生血红素加氧酶-1(heme oxygenase-1,HO-1)来发挥对缺血再灌注损伤的保护作用,并且有研究表明HO-1降解血红素产生的代谢产物一氧化碳(carbon monoxide,CO)也有同样的作用。结论枯否细胞在肝移植术后可以发挥双向性作用,如何减少枯否细胞释放各种有害物质,增加有利物质的表达,是今后预防移植肝后缺血再灌注损伤研究的关键。     

Beneficial Effects of Hydrogen Gas on Porcine Liver Reperfusion Injury With Use of Total Vascular Exclusion and Active Venous Bypass

Background

Liver ischemia/reperfusion (I/R) injury is a high risk factor in liver transplantation and it influences graft survival. One of the major events during I/R injury is the generation of cytotoxic oxygen radicals. Recently, hydrogen gas has been reported to have antioxidant properties and protective effects against organ dysfunction induced by I/R injury. The aim of this study is to investigate effects of hydrogen on porcine liver reperfusion injury.

Materials and Methods

Six outbred pigs weighing 20 kg were used for the experiment. Under general anesthesia, the venous bypass between the left femoral vein and the splenic vein to the left jugular vein was made using a centrifugal pump. Then, we used a total vascular exclusion clamp (all in- and out-flow to the liver was clamped) for 60 minutes. Hydrogen (5 ppm) saturated with lactate Ringer's solution was prepared. This solution was infused through the portal vein just before reperfusion (hydrogen group).

Results

Aspartate aminotransferase levels in the control versus hydrogen group in 30, 60, and 120 minutes after reperfusion were 1560.3, 1925.3, and 2342.5 versus 175.3, 200.7, and 661.00 IU/L, respectively. Lactate dehydrogenase (LDH) levels in the control versus hydrogen groups in 30, 60, and 120 minutes after reperfusion were 23,235.0, 3496.7, and 4793.5 versus 663.3, 802.0, and 983.7 IU/L, respectively. The hydrogen gas level in liver tissue increased to 954.6 ppm immediately after reperfusion; however, it disappeared within 30 minutes.

Conclusion

The solution containing hydrogen gas was safe and had remarkably protective effects on the porcine during liver I/R and may be applied in the clinical setting.