Corrigendum to "Release of Danger Signals during Ischemic Storage of the Liver: A Potential Marker of Organ Damage?"

[This corrects the article DOI: 10.1155/2010/436145.].

Nondestructive molecular imaging by Raman spectroscopy vs. marker detection by MALDI IMS for an early diagnosis of HCC.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide with a steadily increasing mortality rate. Fast diagnosis at early stages of HCC is of key importance for the improvement of patient survival rates. In this regard, we combined two imaging techniques with high potential for HCC diagnosis in order to improve the prediction of liver cancer. In detail, Raman spectroscopic imaging and matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) were applied for the diagnosis of 36 HCC tissue samples. The data were analyzed using multivariate methods, and the results revealed that Raman spectroscopy alone showed a good capability for HCC tumor identification (sensitivity of 88% and specificity of 80%), which could not be improved by combining the Raman data with MALDI IMS. In addition, it could be shown that the two methods in combination can differentiate between well-, moderately- and poorly-differentiated HCC using a linear classification model. MALDI IMS not only classified the HCC grades with a sensitivity of 100% and a specificity of 80%, but also showed significant differences in the expression of glycerophospholipids and fatty acyls during HCC differentiation. Furthermore, important differences in the protein, lipid and collagen compositions of differentiated HCC were detected using the model coefficients of a Raman based classification model. Both Raman and MALDI IMS, as well as their combination showed high potential for resolving concrete questions in liver cancer diagnosis.

Young plasma attenuates age-dependent liver ischemia reperfusion injury.

Aging is often associated with a decreased autophagic activity that contributes to the high sensitivity of aged livers to ischemia reperfusion injury (IRI). Blood from young animals can positively affect aged animals. This study was designed to evaluate the effect of young plasma in a model of liver IRI in aged rats. Aged rats were treated with pooled plasma collected from young rats before ischemia. Administration of young plasma restored aging-induced suppression in hepatic autophagic activity and reduced liver IRI. Inhibition of the young-plasma-restored autophagic activity abrogated the beneficial effect of young plasma against liver IRI. Similarly, young serum restored autophagic activity and reduced cellular injury after hypoxia/reoxygenation (H/R) in primary old rat hepatocytes. Mechanistic studies showed thatadministration of young plasma increased AMPK phosphorylation and led to unc-51-like autophagy activating kinase (ULK)1 activation. Furthermore, AMPK-inhibition abrogated the young serum-induced ULK1 activation and autophagic activity and diminished the protective action of young serum against H/R injury in primary old rat hepatocytes, whereas AMPK-activation potentiated the effects of young serum. Young plasma could restore age-impaired autophagy, at least in part, via AMPK/ULK1 signaling. Restoration of age-impaired autophagic activity may be a critical contributing mechanism to young-plasma-afforded protection against liver IRI in aged rats.-Liu, A., Yang, J., Hu, Q., Dirsch, O., Dahmen, U., Zhang, C., Gewirtz, D. A., Fang, H., Sun, J. Young plasma attenuates age-dependent liver ischemia reperfusion injury.

The Role of Autophagy for the Regeneration of the Aging Liver.

Age is one of the key risk factors to develop malignant diseases leading to a high incidence of hepatic tumors in the elderly population. The only curative treatment for hepatic tumors is surgical removal, which initiates liver regeneration. However, liver regeneration is impaired with aging, leading to an increased surgical risk for the elderly patient. Due to the increased risk, those patients are potentially excluded from curative surgery. Aging impairs autophagy via lipofuscin accumulation and inhibition of autophagosome formation. Autophagy is a recycling mechanism for eukaryotic cells to maintain homeostasis. Its principal function is to degrade endogenous bio-macromolecules for recycling cellular substances. A number of recent studies have shown that the reduced regenerative capacity of the aged remnant liver can be restored by promoting autophagy. Autophagy can be activated via multiple mTOR-dependent and mTOR-independent pathways. However, inducing autophagy through the mTOR-dependent pathway alone severely impairs liver regeneration. In contrast, recent observations suggest that inducing autophagy via mTOR-independent pathways might be promising in promoting liver regeneration. Conclusion: Activation of autophagy via an mTOR-independent autophagy inducer is a potential therapy for promoting liver regeneration, especially in the elderly patients at risk.

Growth differentiation factor 11 worsens hepatocellular injury and liver regeneration after liver ischemia reperfusion injury.

Growth differentiation factor 11 (GDF11) has been implicated in a variety of aging conditions and the regulation of organ regeneration after injury; however, the role of GDF11 in liver ischemia reperfusion injury (IRI) is unknown. The aim of the current study was to investigate the possible role of GDF11 in liver IRI. We investigated the effects of GDF11 in liver IRI in both young (3 mo) and old (22 mo) mice in vivo, and in primary young and old mouse hepatocytes in vitro. Both serum and hepatic GDF11 protein expression levels increased with age and after IRI. Treatment with recombinant GDF11 significantly increased IRI-induced elevations of serum aminotransferase levels, worsened the histologic status of livers, and impaired liver regeneration. In contrast, inhibition of GDF11 activity with neutralizing Abs significantly decreased liver injury and improved liver regeneration after IRI. In vitro, treatment with recombinant GDF11 significantly delayed cell proliferation in cultured hepatocytes that were subjected to hypoxia/reoxygenation insult. Moreover, suppression of cell-cycle progression may be a key mechanism by which GDF11 inhibited hepatocyte regeneration. Collectively, rather than acting as a rejuvenating agent, GDF11 worsens hepatocellular injury and impairs liver regeneration after IRI.-Liu, A., Dong, W., Peng, J., Dirsch, O., Dahmen, U., Fang, H., Zhang, C., Sun, J. Growth differentiation factor 11 worsens hepatocellular injury and liver regeneration after liver ischemia reperfusion injury.

Carbon monoxide ameliorates hepatic ischemia/reperfusion injury via sirtuin 1-mediated deacetylation of high-mobility group box 1 in rats.

Carbon monoxide (CO) exerts protective effects on hepatic ischemia/reperfusion injury (IRI), but the underlying molecular mechanisms are not fully understood. High-mobility group box 1 (HMGB1) is an important mediator of injury and inflammation in hepatic IRI. Here, we investigated whether CO could attenuate hepatic IRI via inhibition of HMGB1 release, particularly through sirtuin 1 (SIRT1). CO was released by treatment with carbon monoxide-releasing molecule (CORM)-2. CORM-2-delivered CO ameliorated hepatic IRI, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory responses, and less severe ischemia/reperfusion-associated histopathologic changes. Treatment with CORM-2 significantly inhibited IRI-induced HMGB1 translocation and release. SIRT1 expression was increased by CORM-2 pretreatment. When CORM-2-induced SIRT1 expression was inhibited using EX527, HMGB1 translocation and release were increased and hepatic IRI was worsened, whereas SIRT1 activation by resveratrol reversed this trend. In vitro, CORM-2 reduced hypoxia/reoxygenation-induced HMGB1 translocation and release, these inhibitions were blocked by SIRT1 inhibition using EX527 or SIRT1 small interfering RNA both in alpha mouse liver 12 cells and RAW264.7 macrophages. Moreover, SIRT1 directly interacted with and deacetylated HMGB1. IRI increased HMGB1 acetylation, which was abolished by CORM-2 treatment via SIRT1. In conclusion, these results suggest that CO may increase SIRT1 expression, which may decrease HMGB1 acetylation and subsequently reduce its translocation and release, thereby protecting against hepatic IRI. Liver Transplantation 23 510-526 2017 AASLD.

Cholestasis-induced adaptive remodeling of interlobular bile ducts.

UNLABELLED: Cholestasis is a common complication in liver diseases that triggers a proliferative response of the biliary tree. Bile duct ligation (BDL) is a frequently used model of cholestasis in rodents. To determine which changes occur in the three-dimensional (3D) architecture of the interlobular bile duct during cholestasis, we used 3D confocal imaging, surface reconstructions, and automated image quantification covering a period up to 28 days after BDL. We show a highly reproducible sequence of interlobular duct remodeling, where cholangiocyte proliferation initially causes corrugation of the luminal duct surface, leading to an approximately five-fold increase in surface area. This is analogous to the function of villi in the intestine or sulci in the brain, where an expansion of area is achieved within a restricted volume. The increase in surface area is further enhanced by duct branching, branch elongation, and loop formation through self-joining, whereby an initially relatively sparse mesh surrounding the portal vein becomes five-fold denser through elongation, corrugation, and ramification. The number of connections between the bile duct and the lobular bile canalicular network by the canals of Hering decreases proportionally to the increase in bile duct length, suggesting that no novel connections are established. The diameter of the interlobular bile duct remains constant after BDL, a response that is qualitatively distinct from that of large bile ducts, which tend to enlarge their diameters. Therefore, volume enhancement is only due to net elongation of the ducts. Because curvature and tortuosity of the bile duct are unaltered, this enlargement of the biliary tree is caused by branching and not by convolution. CONCLUSION: BDL causes adaptive remodeling that aims at optimizing the intraluminal surface area by way of corrugation and branching.

Somatostatin and CXCR4 chemokine receptor expression in hepatocellular and cholangiocellular carcinomas: tumor capillaries as promising targets.

BACKGROUND: Hepatocellular (HCC) and cholangiocellular carcinomas (CCC) display an exceptionally poor prognosis. Especially for advanced disease no efficient standard therapy is currently available. Recently, somatostatin analogs have been evaluated for the treatment of HCC, however, with contradictory results. Besides, for both malignancies the chemokine receptor CXCR4 has been discussed as a possible new target structure. METHODS: Expression of somatostatin receptor (SSTR) subtypes 1, 2A, 3, 4, and 5, and of CXCR4 was evaluated in a total of 71 HCCs and 27 CCCs by immunohistochemistry using well-characterized novel monoclonal antibodies. RESULTS: In HCC tumor cells, frequency and intensity of expression of SSTRs and CXCR4 were only low. CXCR4 was present in about 40% of the HCCs, although at a low intensity. SSTR5, SSTR2, and SSTR3 were detected in about 15%, 8%, and 5% of the HCC tumors, respectively. SSTR and CXCR4 expression was much higher in CCC than in HCC. CXCR4 and SSTR1 were present in 60% and 67% of the CCC samples, respectively, followed by SSTR2 and SSTR5, which were detected in 30% and 11% of the tumors, respectively. Most notably, CXCR4 was intensely expressed on the tumor capillaries in about 50% of the HCCs and CCCs. CXCR4 expression on tumor vessels was associated with poor patient outcomes. CONCLUSIONS: CCC, but not HCC, may be suitable for SSTR-based treatments. Because of the predominant expression of SSTR1, pan-somatostatin analogs should be preferred. In both HCC and CCC, indirect targeting of tumors via the CXCR4-positive tumor capillaries may represent a promising additional therapeutic strategy.

Induction of autophagy reduces ischemia/reperfusion injury in steatotic rat livers.

BACKGROUND: Steatotic livers are particularly vulnerable to ischemia/reperfusion injury (IRI). One of the reasons is an underlying impairment of autophagy. Autophagy is regulated by glycogen synthase kinase 3b (GSK3b) and extracellular signal-regulated kinases (ERK1/2) pathways. Both of them are target proteins of a cell-protective drug, lithium chloride. Lithium chloride treatment reduces IRI in many organs including liver. Therefore, we aimed to investigate the effect of lithium chloride treatment on autophagy induction in steatotic rat livers. We also wanted to evaluate the related cell-protective effects on the enhanced hepatic IRI. MATERIALS AND METHODS: After inducing hepatic steatosis, rats were injected with lithium chloride or normal saline for 3 d before being subjected to 70% selective warm ischemia for 60 min. After reperfusion, rats were observed for 30 min, 6, 24, and 48 h. RESULTS: Lithium chloride appeared to protect hepatocytes from IRI via its ability to induce autophagy by modulation of both GSK3b and ERK1/2 pathways. Hepatic damage was significantly decreased in the treatment group as indicated by a reduced inflammatory response, less apoptosis, less necrosis, and lower liver enzyme levels. CONCLUSIONS: Simultaneous modulation of GSK3b and ERK1/2 pathways might be an interesting strategy to reduce IRI in steatotic livers with an impairment of autophagy.

Identification of Proteins Interacting with Cytoplasmic High-Mobility Group Box 1 during the Hepatocellular Response to Ischemia Reperfusion Injury.

Ischemia/reperfusion injury (IRI) occurs inevitably in liver transplantations and frequently during major resections, and can lead to liver dysfunction as well as systemic disorders. High-mobility group box 1 (HMGB1) plays a pathogenic role in hepatic IRI. In the normal liver, HMGB1 is located in the nucleus of hepatocytes; after ischemia reperfusion, it translocates to the cytoplasm and it is further released to the extracellular space. Unlike the well-explored functions of nuclear and extracellular HMGB1, the role of cytoplasmic HMGB1 in hepatic IRI remains elusive. We hypothesized that cytoplasmic HMGB1 interacts with binding proteins involved in the hepatocellular response to IRI. In this study, binding proteins of cytoplasmic HMGB1 during hepatic IRI were identified. Liver tissues from rats with warm ischemia reperfusion (WI/R) injury and from normal rats were subjected to cytoplasmic protein extraction. Co-immunoprecipitation using these protein extracts was performed to enrich HMGB1-protein complexes. To separate and identify the immunoprecipitated proteins in eluates, 2-dimensional electrophoresis and subsequent mass spectrometry detection were performed. Two of the identified proteins were verified using Western blotting: betaine-homocysteine S-methyltransferase 1 (BHMT) and cystathionine γ-lyase (CTH). Therefore, our results revealed the binding of HMGB1 to BHMT and CTH in cytoplasm during hepatic WI/R. This finding may help to better understand the cellular response to IRI in the liver and to identify novel molecular targets for reducing ischemic injury.

Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue.

It is well known that isolation and cultivation of primary hepatocytes cause major gene expression alterations. In the present genome-wide, time-resolved study of cultivated human and mouse hepatocytes, we made the observation that expression changes in culture strongly resemble alterations in liver diseases. Hepatocytes of both species were cultivated in collagen sandwich and in monolayer conditions. Genome-wide data were also obtained from human NAFLD, cirrhosis, HCC and hepatitis B virus-infected tissue as well as mouse livers after partial hepatectomy, CCl4 intoxication, obesity, HCC and LPS. A strong similarity between cultivation and disease-induced expression alterations was observed. For example, expression changes in hepatocytes induced by 1-day cultivation and 1-day CCl4 exposure in vivo correlated with R = 0.615 (p < 0.001). Interspecies comparison identified predominantly similar responses in human and mouse hepatocytes but also a set of genes that responded differently. Unsupervised clustering of altered genes identified three main clusters: (1) downregulated genes corresponding to mature liver functions, (2) upregulation of an inflammation/RNA processing cluster and (3) upregulated migration/cell cycle-associated genes. Gene regulatory network analysis highlights overrepresented and deregulated HNF4 and CAR (Cluster 1), Krüppel-like factors MafF and ELK1 (Cluster 2) as well as ETF (Cluster 3) among the interspecies conserved key regulators of expression changes. Interventions ameliorating but not abrogating cultivation-induced responses include removal of non-parenchymal cells, generation of the hepatocytes' own matrix in spheroids, supplementation with bile salts and siRNA-mediated suppression of key transcription factors. In conclusion, this study shows that gene regulatory network alterations of cultivated hepatocytes resemble those of inflammatory liver diseases and should therefore be considered and exploited as disease models.

Attitude towards organ donation in German medical students.

PURPOSE: It is well known that personal decision making in respect to organ donation is highly dependent on the balance of knowledge, trust, and fear. We wanted to explore the attitude of German medical students towards organ donation and investigate the relationship between knowledge, trust, and fear in this special subgroup. METHODS: We conducted an online survey utilizing (1) the snowball effect of using Facebook groups and advertisement as well as (2) mailing lists of medical faculties in Germany for distribution. RESULTS: We surveyed 1370 medical students. 75.8 % (N = 988) of the participants stated to carry an organ donor card and allowed their organs to be donated. 1.8 % (N = 23) refused donation. 22.5 % (N = 293) did not carry an organ donor card. Analysis of the "decided" versus the "undecided" group revealed substantial differences regarding transplantation knowledge (mean knowledge score of 4.23 vs. 3.81; P < 0.001), trust in (mean trust score 4.11 vs. 3.39; P < 0.001), and fear of (mean fear score 1.63 vs. 2.22; P < 0.001) organ donation. 45.9 % of the undecided group (N = 134) opted for accessing additional information material. After reading the info material, 22.7 % (N = 29) stated their willingness to sign a donor card, whereas 76.6 % (N = 98) still could not reach a decision. CONCLUSIONS: The willingness to potentially act as organ donor was related to the pre-existent knowledge, trust, and fear. Access to information material did promote the decision towards organ donation in a group of previously undecided medical students. This advocates initiating information campaigns even in population groups with strong medical background.

Bioengineered Livers: A New Tool for Drug Testing and a Promising Solution to Meet the Growing Demand for Donor Organs.

BACKGROUND: Organ engineering is a new innovative strategy to cope with two problems: the need for physiological models for pharmacological research and donor organs for transplantation. A functional scaffold is generated from explanted organs by removing all cells (decellularization) by perfusing the organ with ionic or nonionic detergents via the vascular system. Subsequently the acellular scaffold is reseeded with organ-specific cells (repopulation) to generate a functional organ. SUMMARY: This review gives an overview of the state of the art describing the decellularization process, the subsequent quality assessment, the repopulation techniques and the functional assessment. It emphasizes the use of scaffolds as matrix for culturing human liver cells for drug testing. Further, it highlights the techniques for transplanting these engineered scaffolds in allogeneic or xenogeneic animals in order to test their biocompatibility and use as organ grafts. Key Messages: The first issue is the so-called decellularization, which is best explored and resulted in a multitude of different protocols. The most promising approach seems to be the combination of pulsatile perfusion of the liver with Triton X-100 and SDS via hepatic artery and portal vein. Widely accepted parameters of quality control include the quantitative assessment of the DNA content and the visualization of eventually remaining nuclei confirmed by HE staining. Investigations regarding the composition of the extracellular matrix focused on histological determination of laminin, collagen, fibronectin and elastin and remained qualitatively. Repopulation is the second issue which is addressed. Selection of the most suitable cell type is a highly controversial topic. Currently, the highest potential is seen for progenitor and stem cells. Cells are infused into the scaffold and cultured under static conditions or in a bioreactor allowing dynamic perfusion of the scaffold. The quality of repopulation is mainly assessed by routine histology and basic functional assays. These promising results prompted to consider the use of a liver scaffold repopulated with human cells for pharmacological research. Transplantation of the (repopulated) scaffold is the third topic which is not yet widely addressed. Few studies report the heterotopic transplantation of repopulated liver tissue without vascular anastomosis. Even fewer studies deal with the heterotopic transplantation of a scaffold or a repopulated liver lobe. However, observation time was still limited to hours, and long-term graft survival has not been reported yet. These exciting results emphasize the potential of this new and promising strategy to create physiological models for pharmacological research and to generate liver grafts for the transplant community to treat organ failure. However, the scientific need for further development in the field of liver engineering is still tremendous.

Intrahepatic Size Regulation in a Surgical Model: Liver Resection-Induced Liver Regeneration Counteracts the Local Atrophy following Simultaneous Portal Vein Ligation.

BACKGROUND/AIM: Liver size regulation is based on the balance between hepatic regeneration and atrophy. To achieve a better understanding of intrahepatic size regulation, we explored the size regulation of a portally deprived liver lobe on a liver subjected to concurrent portal vein ligation (PVL) and partial hepatectomy (PHx). MATERIALS AND METHODS: Using a surgical rat model consisting of right PVL (rPVL) plus 70% PHx, we evaluated the size regulation of liver lobes 1, 2, 3, and 7 days after the operation in terms of liver weight and hepatocyte proliferation. Portal hyperperfusion was confirmed by measuring portal flow. The portal vascular tree was visualized by injection of a contrast agent followed by CT imaging of explanted livers. Control groups consisted of 70% PHx, rPVL, and sham operation. RESULTS: The size of the ligated right lobe increased to 1.4-fold on postoperative day 7 when subjected to rPVL + 70% PHx. The right lobe increased to 3-fold when subjected to 70% PHx alone and decreased to 0.3-fold when subjected to rPVL only. The small but significant increase in liver weight after the combined procedure was accompanied by a low proliferative response. In contrast, hepatocyte proliferation was undetectable in the right lobe undergoing atrophy after PVL only. The caudate lobe in the rPVL + 70% PHx group increased to 4.6-fold, which is significantly more than in the other groups. This increase in liver weight was paralleled by persisting portal hyperperfusion and a prolonged proliferative phase of 3 days. CONCLUSIONS: A discontinued portal blood supply does not always result in atrophy of the ligated lobe. The concurrent regenerative stimulus induced by 70% PHx seemed to counteract the local atrophy after a simultaneously performed rPVL, leading to a low but prolonged regenerative response of the portally deprived liver lobe. This observation supports the conclusion that portal flow is not necessary for liver regeneration. The persisting portal hyperperfusion may be crucial for the specific kinetics of prolonged liver regeneration after rPVL + 70% PHx in the portally supplied caudate lobe. Both observations deserve more attention regarding the underlying mechanism in further studies.

Novel Woodchuck Hepatitis Virus (WHV) transgene mouse models show sex-dependent WHV replicative activity and development of spontaneous immune responses to WHV proteins.

The woodchuck model is an informative model for studies on hepadnaviral infection. In this study, woodchuck hepatitis virus (WHV) transgenic (Tg) mouse models based on C57BL/6 mice were established to study the pathogenesis associated with hepadnaviral infection. Two lineages of WHV Tg mice, harboring the WHV wild-type genome (lineage 1217) and a mutated WHV genome lacking surface antigen (lineage 1281), were generated. WHV replication intermediates were detected by Southern blotting. DNA vaccines against WHV proteins were applied by intramuscular injection. WHV-specific immune responses were analyzed by flow cytometry and enzyme-linked immunosorbent assays (ELISAs). The presence of WHV transgenes resulted in liver-specific but sex- and age-dependent WHV replication in Tg mice. Pathological changes in the liver, including hepatocellular dysplasia, were observed in aged Tg mice, suggesting that the presence of WHV transgenes may lead to liver diseases. Interestingly, Tg mice of lineage 1281 spontaneously developed T- and B-cell responses to WHV core protein (WHcAg). DNA vaccination induced specific immune responses to WHV proteins in WHV Tg mice, indicating a tolerance break. The magnitude of the induced WHcAg-specific immune responses was dependent on the effectiveness of different DNA vaccines and was associated with a decrease in WHV loads in mice. In conclusion, sex- and age-dependent viral replication, development of autoimmune responses to viral antigens, pathological changes in the liver in WHV Tg mice, and the possibility of breaking immune tolerance to WHV transgenes will allow future studies on pathogenesis related to hepadnaviral infection and therapeutic vaccines.

Rodent models and imaging techniques to study liver regeneration.

The liver has the unique capability of regeneration from various injuries. Different animal models and in vitro methods are used for studying the processes and mechanisms of liver regeneration. Animal models were established either by administration of hepatotoxic chemicals or by surgical approach. The administration of hepatotoxic chemicals results in the death of liver cells and in subsequent hepatic regeneration and tissue repair. Surgery includes partial hepatectomy and portal vein occlusion or diversion: hepatectomy leads to compensatory regeneration of the remnant liver lobe, whereas portal vein occlusion leads to atrophy of the ipsilateral lobe and to compensatory regeneration of the contralateral lobe. Adaptation of modern radiological imaging technologies to the small size of rodents made the visualization of rodent intrahepatic vascular anatomy possible. Advanced knowledge of the detailed intrahepatic 3D anatomy enabled the establishment of refined surgical techniques. The same technology allows the visualization of hepatic vascular regeneration. The development of modern histological image analysis tools improved the quantitative assessment of hepatic regeneration. Novel image analysis tools enable us to quantify reliably and reproducibly the proliferative rate of hepatocytes using whole-slide scans, thus reducing the sampling error. In this review, the refined rodent models and the newly developed imaging technology to study liver regeneration are summarized. This summary helps to integrate the current knowledge of liver regeneration and promises an enormous increase in hepatological knowledge in the near future.

Ischemic preconditioning protects against liver ischemia/reperfusion injury via heme oxygenase-1-mediated autophagy.

OBJECTIVES: Ischemic preconditioning exerts a protective effect in hepatic ischemia/reperfusion injury. The exact mechanism of ischemic preconditioning action remains largely unknown. Recent studies suggest that autophagy plays an important role in protecting against ischemia/reperfusion injury. However, the role of autophagy in ischemic preconditioning-afforded protection and its regulatory mechanisms in liver ischemia/reperfusion injury remain poorly understood. This study was designed to determine whether ischemic preconditioning could protect against liver ischemia/reperfusion injury via heme oxygenase-1-mediated autophagy. DESIGN: Laboratory investigation. SETTING: University animal research laboratory. SUBJECTS: Male inbred Lewis rats and C57BL/6 mice. INTERVENTIONS: Ischemic preconditioning was produced by 10 minutes of ischemia followed by 10 minutes of reperfusion prior to 60 minutes of ischemia. In a rat model of hepatic ischemia/reperfusion injury, rats were pretreated with wortmannin or rapamycin to evaluate the contribution of autophagy to the protective effects of ischemic preconditioning. Heme oxygenase-1 was inhibited with tin protoporphyrin IX. In a mouse model of hepatic ischemia/reperfusion injury, autophagy or heme oxygenase-1 was inhibited with vacuolar protein sorting 34 small interfering RNA or heme oxygenase-1 small interfering RNA, respectively. MEASUREMENTS AND MAIN RESULTS: Ischemic preconditioning ameliorated liver ischemia/reperfusion injury, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory cytokines, and less severe ischemia/reperfusion-associated histopathologic changes. Ischemic preconditioning treatment induced autophagy activation, as indicated by an increase of LC3-II, degradation of p62, and accumulation of autophagic vacuoles in response to ischemia/reperfusion injury. When ischemic preconditioning-induced autophagy was inhibited with wortmannin in rats or vacuolar protein sorting 34-specific small interfering RNA in mice, liver ischemia/reperfusion injury was worsened, whereas rapamycin treatment increased autophagy and mimicked the protective effects of ischemic preconditioning. Furthermore, ischemic preconditioning increased heme oxygenase-1 expression. The inhibition of heme oxygenase-1 with tin protoporphyrin IX in rats or heme oxygenase-1-specific small interfering RNA in mice decreased ischemic preconditioning-induced autophagy and diminished the protective effects of ischemic preconditioning against ischemia/reperfusion injury. CONCLUSIONS: Ischemic preconditioning protects against liver ischemia/reperfusion injury, at least in part, via heme oxygenase-1-mediated autophagy.

G-CSF pretreatment aggravates LPS-associated microcirculatory dysfunction and acute liver injury after partial hepatectomy in rats.

Liver dysfunction is a serious complication in the early phase following major liver resection or liver transplantation and might be aggravated by the translocation of bacteria and lipopolysaccharide (LPS). As a preventive strategy, granulocyte colony-stimulating factor (G-CSF) is prophylactically applied in patients who are subjected to major surgery. However, we previously demonstrated that G-CSF can induce LPS sensitization. In this study, we aimed to evaluate the effects of G-CSF pretreatment on hepatic microcirculatory disturbances and postoperative liver dysfunction after 70 % partial hepatectomy (PH) in rats. PH alone was well tolerated by all animals (100 % survival rate, slight liver damage and inflammation). LPS application after 70 % PH caused moderate inflammation, microcirculatory disturbances and hepatic damage and led to a 24-h survival rate of 30 % after the operations. In the G-CSF-LPS-PH group, all of the rats died within 4 h with severe inflammatory responses and liver damage (i.e., pronounced erythrocyte congestion and neutrophil infiltration). Portal hypertension and microcirculatory disorders (i.e., inhomogeneous perfusion, sinusoidal dilatation and reductions on functional capillary density) were more pronounced in the G-CSF-LPS-PH group. In conclusion, increased circulating LPS levels were associated with an imbalanced inflammatory response and microcirculatory dysfunction that preceded liver damage and subsequent dysfunction following surgery. G-CSF-pretreatment aggravated microcirculatory disturbances and liver damage, which might have been related to G-CSF-induced LPS sensitization.

Protocols for staining of bile canalicular and sinusoidal networks of human, mouse and pig livers, three-dimensional reconstruction and quantification of tissue microarchitecture by image processing and analysis.

Histological alterations often constitute a fingerprint of toxicity and diseases. The extent to which these alterations are cause or consequence of compromised organ function, and the underlying mechanisms involved is a matter of intensive research. In particular, liver disease is often associated with altered tissue microarchitecture, which in turn may compromise perfusion and functionality. Research in this field requires the development and orchestration of new techniques into standardized processing pipelines that can be used to reproducibly quantify tissue architecture. Major bottlenecks include the lack of robust staining, and adequate reconstruction and quantification techniques. To bridge this gap, we established protocols employing specific antibody combinations for immunostaining, confocal imaging, three-dimensional reconstruction of approximately 100-µm-thick tissue blocks and quantification of key architectural features. We describe a standard procedure termed 'liver architectural staining' for the simultaneous visualization of bile canaliculi, sinusoidal endothelial cells, glutamine synthetase (GS) for the identification of central veins, and DAPI as a nuclear marker. Additionally, we present a second standard procedure entitled 'S-phase staining', where S-phase-positive and S-phase-negative nuclei (stained with BrdU and DAPI, respectively), sinusoidal endothelial cells and GS are stained. The techniques include three-dimensional reconstruction of the sinusoidal and bile canalicular networks from the same tissue block, and robust capture of position, size and shape of individual hepatocytes, as well as entire lobules from the same tissue specimen. In addition to the protocols, we have also established image analysis software that allows relational and hierarchical quantifications of different liver substructures (e.g. cells and vascular branches) and events (e.g. cell proliferation and death). Typical results acquired for routinely quantified parameters in adult mice (C57Bl6/N) include the hepatocyte volume (5,128.3 ± 837.8 µm(3)) and the fraction of the hepatocyte surface in contact with the neighbouring hepatocytes (67.4 ± 6.7 %), sinusoids (22.1 ± 4.8 %) and bile canaliculi (9.9 ± 3.8 %). Parameters of the sinusoidal network that we also routinely quantify include the radius of the sinusoids (4.8 ± 2.25 µm), the branching angle (32.5 ± 11.2°), the length of intersection branches (23.93 ± 5.9 µm), the number of intersection nodes per mm(3) (120.3 × 103 ± 42.1 × 10(3)), the average length of sinusoidal vessel per mm(3) (5.4 × 10(3) ± 1.4 × 10(3)mm) and the percentage of vessel volume in relation to the whole liver volume (15.3 ± 3.9) (mean ± standard deviation). Moreover, the provided parameters of the bile canalicular network are: length of the first-order branches (7.5 ± 0.6 µm), length of the second-order branches (10.9 ± 1.8 µm), length of the dead-end branches (5.9 ± 0.7 µm), the number of intersection nodes per mm(3) (819.1 × 10(3) ± 180.7 × 10(3)), the number of dead-end branches per mm(3) (409.9 × 10(3) ± 95.6 × 10(3)), the length of the bile canalicular network per mm(3) (9.4 × 10(3) ± 0.7 × 10(3) mm) and the percentage of the bile canalicular volume with respect to the total liver volume (3.4 ± 0.005). A particular strength of our technique is that quantitative parameters of hepatocytes and bile canalicular as well as sinusoidal networks can be extracted from the same tissue block. Reconstructions and quantifications performed as described in the current protocols can be used for quantitative mathematical modelling of the underlying mechanisms. Furthermore, protocols are presented for both human and pig livers. The technique is also applicable for both vibratome blocks and conventional paraffin slices.