[Surprising diagnosis after suspected appendicitis]. / Verrassende diagnose na verdenking appendicitis.

A 12-year-old boy presented at the emergency department because of right-sided abdominal pain. Laboratory findings and ultrasound examination were suggestive of acute appendicitis. During laparoscopy, an indurated omental mass was seen. The appendix was normal. Histopathological examination confirmed a diagnosis of omental infarction, which is rare in pediatric patients.

Experimental Conditions That Influence the Utility of 2'7'-Dichlorodihydrofluorescein Diacetate (DCFH2-DA) as a Fluorogenic Biosensor for Mitochondrial Redox Status.

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2',7'-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2',7'-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2',7'-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol.

Optimal Use of 2',7'-Dichlorofluorescein Diacetate in Cultured Hepatocytes.

Oxidative stress is a state that arises when the production of reactive transients overwhelms the cell's capacity to neutralize the oxidants and radicals. This state often coincides with the pathogenesis and perpetuation of numerous chronic diseases. On the other hand, medical interventions such as radiation therapy and photodynamic therapy generate radicals to selectively damage and kill diseased tissue. As a result, the qualification and quantification of oxidative stress are of great interest to those studying disease mechanisms as well as therapeutic interventions. 2',7'-Dichlorodihydrofluorescein-diacetate (DCFH2-DA) is one of the most widely used fluorogenic probes for the detection of reactive transients. The nonfluorescent DCFH2-DA crosses the plasma membrane and is deacetylated by cytosolic esterases to 2',7'-dichlorodihydrofluorescein (DCFH2). The nonfluorescent DCFH2 is subsequently oxidized by reactive transients to form the fluorescent 2',7'-dichlorofluorescein (DCF). The use of DCFH2-DA in hepatocyte-derived cell lines is more challenging because of membrane transport proteins that interfere with probe uptake and retention, among several other reasons. Cancer cells share some of the physiological and biochemical features with hepatocytes, so probe-related technical issues are applicable to cultured malignant cells as well. This study therefore analyzed the in vitro properties of DCFH2-DA in cultured human hepatocytes (HepG2 cells and differentiated and undifferentiated HepaRG cells) to identify methodological and technical features that could impair proper data analysis and interpretation. The main issues that were found and should therefore be accounted for in experimental design include the following: (1) both DCFH2-DA and DCF are taken up rapidly, (2) DCF is poorly retained in the cytosol and exits the cell, (3) the rate of DCFH2 oxidation is cell type-specific, (4) DCF fluorescence intensity is pH-dependent at pH < 7, and (5) the stability of DCFH2-DA in cell culture medium relies on medium composition. Based on the findings, the conditions for the use of DCFH2-DA in hepatocyte cell lines were optimized. Finally, the optimized protocol was reduced to practice and DCFH2-DA was applied to visualize and quantify oxidative stress in real time in HepG2 cells subjected to anoxia/reoxygenation as a source of reactive transients.

Analysis and Optimization of Conditions for the Use of 2',7'-Dichlorofluorescein Diacetate in Cultured Hepatocytes.

Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2',7'-dichlorodihydrofluorescein-diacetate (DCFH2-DA) as fluorogenic redox probe is problematic in liver cell lines because of membrane transport proteins that interfere with probe kinetics, among other reasons. The properties of DCFH2-DA were analyzed in hepatocytes (HepG2, HepaRG) to characterize methodological issues that could hamper data interpretation and falsely skew conclusions. Experiments were focused on probe stability in relevant media, cellular probe uptake/retention/excretion, and basal oxidant formation and metabolism. DCFH2-DA was used under optimized experimental conditions to intravitally visualize and quantify oxidative stress in real-time in HepG2 cells subjected to anoxia/reoxygenation. The most important findings were that: (1) the non-fluorescent DCFH2-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH2 oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH2-DA in cell culture medium relied on medium composition. The use of DCFH2-DA to measure oxidative stress in cultured hepatocytes comes with methodological and technical challenges, which were characterized and solved. Optimized in vitro and intravital imaging protocols were formulated to help researchers conduct proper experiments and draw robust conclusions.

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion.

OBJECTIVE AND BACKGROUND: Activation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice. METHODS: Plasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury. RESULTS: In patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects. CONCLUSION: HMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage.

FXR agonist obeticholic acid induces liver growth but exacerbates biliary injury in rats with obstructive cholestasis.

Cholestasis impairs liver regeneration following partial liver resection (PHx). Bile acid receptor farnesoid X-receptor (FXR) is a key mediator of liver regeneration. The effects of FXR agonist obeticholic acid (OCA) on liver (re)growth were therefore studied in cholestatic rats. Animals underwent sham surgery or reversible bile duct ligation (rBDL). PHx with concurrent internal biliary drainage was performed 7 days after rBDL. Animals were untreated or received OCA (10 mg/kg/day) per oral gavage from rBDL until sacrifice. After 7 days of OCA treatment, dry liver weight increased in the rBDL + OCA group, indicating OCA-mediated liver growth. Enhanced proliferation in the rBDL + OCA group prior to PHx concurred with a rise in Ki67-positive hepatocytes, elevated hepatic Ccnd1 and Cdc25b expression, and an induction of intestinal fibroblast growth factor 15 expression. Liver regrowth after PHx was initially stagnant in the rBDL + OCA group, possibly due to hepatomegaly prior to PHx. OCA increased hepatobiliary injury markers during BDL, which was accompanied by upregulation of the bile salt export pump. There were no differences in histological liver injury. In conclusion, OCA induces liver growth in cholestatic rats prior to PHx but exacerbates biliary injury during cholestasis, likely by forced pumping of bile acids into an obstructed biliary tree.

Hypothermic perfusion with retrograde outflow during right hepatectomy is safe and feasible.

BACKGROUND: In situ hypothermic perfusion during liver resection performed under vascular inflow occlusion decreases hepatic ischemia-reperfusion injury, but technical limitations have restricted its widespread use. In situ hypothermic perfusion with retrograde outflow circumvents these impediments and thus could extend the applicability of in situ hypothermic perfusion. The safety and feasibility of in situ hypothermic perfusion with retrograde outflow were analyzed in selected patients undergoing right (extended) hepatectomy and compared to intermittent vascular inflow occlusion, the gold standard method, in this randomized pilot study. METHODS: Patients were first screened for parenchymal liver disease (exclusion criteria: steatosis ≥30%, cirrhosis, or cholestasis). Study participants were randomized intraoperatively to undergo in situ hypothermic perfusion with retrograde outflow (n = 9) or intermittent vascular inflow occlusion (n = 9). The target liver core temperature during in situ hypothermic perfusion with retrograde outflow was 28°C. The primary end point was ischemia-reperfusion injury (expressed by peak postoperative transaminase levels). Secondary outcomes included functional liver regeneration (assessed by hepatobiliary scintigraphy) and clinical outcomes. RESULTS: Peak transaminase levels, total bilirubin, and the international normalized ratio were similar between both groups, although a trend toward more rapid normalization of bilirubin levels was noted for the in situ hypothermic perfusion with retrograde outflow group. Functional liver regeneration as evaluated by hepatobiliary scintigraphy was improved on postoperative day 3 fafter in situ hypothermic perfusion with retrograde outflow but not after intermittent vascular inflow occlusion. Furthermore, in situ hypothermic perfusion with retrograde outflow (requiring continuous ischemia) was comparable to intermittent vascular inflow occlusion for all clinical outcomes, including postoperative complications and hospital stay. CONCLUSION: The use of in situ hypothermic perfusion with retrograde outflow appears to be safe and feasible in selected patients with healthy liver parenchyma and may benefit early functional liver regeneration. Future applications of in situ hypothermic perfusion with retrograde outflow include patients with damaged liver parenchyma who would require major hepatic resection with a prolonged vascular inflow occlusion duration.

Preparation and Practical Applications of 2',7'-Dichlorodihydrofluorescein in Redox Assays.

Oxidative stress, a state in which intra- or extracellular oxidant production outweighs the antioxidative capacity, lies at the basis of many diseases. DCFH2-DA (2',7'-dichlorodihydrofluorescein diacetate) is the most widely used fluorogenic probe for the detection of general oxidative stress. However, the use of DCFH2-DA, as many other fluorogenic redox probes, is mainly confined to the detection of intracellular oxidative stress in vitro. To expand the applicability of the probe, an alkaline hydrolysis and solvent extraction procedure was developed to generate high-purity DCFH2 (2',7'-dichlorodihydrofluorescein) from DCFH2-DA using basic laboratory equipment. Next, the utility of DCFH2 was exemplified in a variety of cell-free and in vitro redox assay systems, including oxidant production by transition metals, photodynamic therapy, activated macrophages, and platelets, as well as the antioxidative capacity of different antioxidants. In cells, the concomitant use of DCFH2-DA and DCFH2 enabled the measurement and compartmentalized analysis of intra- and extracellularly produced oxidants, respectively, using a single read-out parameter. Furthermore, hepatocyte-targeted liposomes were developed to deliver the carboxylated derivative, 5(6)-carboxy-DCFH2, to hepatocytes in vivo. Liposome-delivered 5(6)-carboxy-DCFH2 enabled real-time visualization and measurement of hepatocellular oxidant production during liver ischemia-reperfusion. The liposomal 5(6)-carboxy-DCFH2 can be targeted to other tissues where oxidative stress is important, including cancer.

How much ischemia can the liver tolerate during resection?

The use of vascular inflow occlusion (VIO, also known as the Pringle maneuver) during liver surgery prevents severe blood loss and the need for blood transfusion. The most commonly used technique for VIO entails clamping of the portal triad, which simultaneously occludes the proper hepatic artery and portal vein. Although VIO is an effective technique to reduce intraoperative blood loss, it also inevitably inflicts hepatic ischemia/reperfusion (I/R) injury as a side effect. I/R injury induces formation of reactive oxygen species that cause oxidative stress and cell death, ultimately leading to a sterile inflammatory response that causes hepatocellular damage and liver dysfunction that can result in acute liver failure in most severe cases. Since the duration of ischemia correlates positively with the severity of liver injury, there is a need to find the balance between preventing severe blood loss and inducing liver damage through the use of VIO. Although research on the maximum duration of hepatic ischemia has intensified since the beginning of the 1980s, there still is no consensus on the tolerable upper limit. Based on the available literature, it is concluded that intermittent and continuous VIO can both be used safely when ischemia times do not exceed 120 min. However, intermittent VIO should be the preferred technique in cases that require >120 min duration of ischemia.

Protective Mechanisms of Hypothermia in Liver Surgery and Transplantation.

Hepatic ischemia/reperfusion (I/R) injury is a side effect of major liver surgery that often cannot be avoided. Prolonged periods of ischemia put a metabolic strain on hepatocytes and limit the tolerable ischemia and preservation times during liver resection and transplantation, respectively. In both surgical settings, temporarily lowering the metabolic demand of the organ by reducing organ temperature effectively counteracts the negative consequences of an ischemic insult. Despite its routine use, the application of liver cooling is predicated on an incomplete understanding of the underlying protective mechanisms, which has limited a uniform and widespread implementation of liver-cooling techniques. This review therefore addresses how hypothermia-induced hypometabolism modulates hepatocyte metabolism during ischemia and thereby reduces hepatic I/R injury. The mechanisms underlying hypothermia-mediated reduction in energy expenditure during ischemia and the attenuation of mitochondrial production of reactive oxygen species during early reperfusion are described. It is further addressed how hypothermia suppresses the sterile hepatic I/R immune response and preserves the metabolic functionality of hepatocytes. Lastly, a summary of the clinical status quo of the use of liver cooling for liver resection and transplantation is provided.

IL-23 and IL-17A are not involved in hepatic/ischemia reperfusion injury in mouse and man.

BACKGROUND: Hepatic ischemia and reperfusion (I/R) is common in liver surgery and transplantation and compromises postoperative liver function. Hepatic I/R injury is characterized by sterile inflammation that contributes to hepatocellular necrosis. Many immune cells and cytokines have been implicated in hepatic I/R injury. However, the role and relevance of IL-23 and IL-17A remains controversial in literature. Aim: To determine whether the IL-23/IL-17A signaling axis is activated in hepatic I/R using a triple-level experimental approach (in vitro, in vivo, and clinical). METHODS: IL-23 and IL-17A were assayed by ELISA in the supernatant fractions of cultured murine (RAW 264.7) macrophages that were activated by supernatant fractions of necrotic cultured mouse (AML12) hepatocytes. Similarly, levels of these cytokines were determined in plasma samples and liver tissue of mice (N = 85) subjected to partial (70%) liver I/R. Finally, IL-23 and IL-17A were assayed in plasma samples obtained from a controlled cohort of liver resection patients who were either subjected to I/R (N = 27) or not (N = 13). RESULTS: Activated macrophages did not produce IL-23 in response to supernatant of necrotic AML12 hepatocytes. IL-23 and IL-17A were not elevated in mice subjected hepatic I/R and were not elevated in serum from patients subjected to I/R during liver resection. CONCLUSION: IL-23 and IL-17A are not involved in hepatic I/R injury in mouse and man. RELEVANCE FOR PATIENTS: If IL-23 and IL-17A were to mediate hepatocellular injury following I/R, these cytokines would constitute potential therapeutic targets. Since this study has revealed that IL-23 and IL-17A do not play a role in hepatic I/R, other pathways and therapeutic targets should be considered when developing modalities aimed at reducing hepatic I/R injury.

Reactive oxygen and nitrogen species in steatotic hepatocytes: a molecular perspective on the pathophysiology of ischemia-reperfusion injury in the fatty liver.

SIGNIFICANCE: Hepatic ischemia-reperfusion (IR) injury results from the temporary deprivation of hepatic blood supply and is a common side effect of major liver surgery (i.e., transplantation or resection). IR injury, which in most severe cases culminates in acute liver failure, is particularly pronounced in livers that are affected by non-alcoholic fatty liver disease (NAFLD). In NAFLD, fat-laden hepatocytes are damaged by chronic oxidative/nitrosative stress (ONS), a state that is acutely exacerbated during IR, leading to extensive parenchymal damage. RECENT ADVANCES: NAFLD triggers ONS via increased (extra)mitochondrial fatty acid oxidation and activation of the unfolded protein response. ONS is associated with widespread protein and lipid (per)oxidation, which reduces the hepatic antioxidative capacity and shifts the intracellular redox status toward an oxidized state. Moreover, activation of the transcription factor peroxisome proliferator-activated receptor α induces expression of mitochondrial uncoupling protein 2, resulting in depletion of cellular energy (ATP) reserves. The reduction in intracellular antioxidants and ATP in fatty livers consequently gives rise to severe ONS and necrotic cell death during IR. CRITICAL ISSUES: Despite the fact that ONS mediates both NAFLD and IR injury, the interplay between the two conditions has never been described in detail. An integrative overview of the pathophysiology of NAFLD that renders steatotic hepatocytes more vulnerable to IR injury is therefore presented in the context of ONS. FUTURE DIRECTIONS: Effective methods should be devised to alleviate ONS and the consequences thereof in NAFLD before surgery in order to improve resilience of fatty livers to IR injury.

The mechanisms and physiological relevance of glycocalyx degradation in hepatic ischemia/reperfusion injury.

SIGNIFICANCE: Hepatic ischemia/reperfusion (I/R) injury is an inevitable side effect of major liver surgery that can culminate in liver failure. The bulk of I/R-induced liver injury results from an overproduction of reactive oxygen and nitrogen species (ROS/RNS), which inflict both parenchymal and microcirculatory damage. A structure that is particularly prone to oxidative attack and modification is the glycocalyx (GCX), a meshwork of proteoglycans and glycosaminoglycans (GAGs) that covers the lumenal endothelial surface and safeguards microvascular homeostasis. ROS/RNS-mediated degradation of the GCX may exacerbate I/R injury by, for example, inducing vasoconstriction, facilitating leukocyte adherence, and directly activating innate immune cells. RECENT ADVANCES: Preliminary experiments revealed that hepatic sinusoids contain a functional GCX that is damaged during murine hepatic I/R and major liver surgery in patients. There are three ROS that mediate GCX degradation: hydroxyl radicals, carbonate radical anions, and hypochlorous acid (HOCl). HOCl converts GAGs in the GCX to GAG chloramides that become site-specific targets for oxidizing and reducing species and are more efficiently fragmented than the parent molecules. In addition to ROS/RNS, the GAG-degrading enzyme heparanase acts at the endothelial surface to shed the GCX. CRITICAL ISSUES: The GCX seems to be degraded during major liver surgery, but the underlying cause remains ill-defined. FUTURE DIRECTIONS: The relative contribution of the different ROS and RNS intermediates to GCX degradation in vivo, the immunogenic potential of the shed GCX fragments, and the role of heparanase in liver I/R injury all warrant further investigation.

Sterile inflammation in hepatic ischemia/reperfusion injury: present concepts and potential therapeutics.

Ischemia and reperfusion (I/R) injury is an often unavoidable consequence of major liver surgery and is characterized by a sterile inflammatory response that jeopardizes the viability of the organ. The inflammatory response results from acute oxidative and nitrosative stress and consequent hepatocellular death during the early reperfusion phase, which causes the release of endogenous self-antigens known as damage-associated molecular patterns (DAMPs). DAMPs, in turn, are indirectly responsible for a second wave of reactive oxygen and nitrogen species (ROS and RNS) production by driving the chemoattraction of various leukocyte subsets that exacerbate oxidative liver damage during the later stages of reperfusion. In this review, the molecular mechanisms underlying hepatic I/R injury are outlined, with emphasis on the interplay between ROS/RNS, DAMPs, and the cell types that either produce ROS/RNS and DAMPs or respond to them. This theoretical background is subsequently used to explain why current interventions for hepatic I/R injury have not been very successful. Moreover, novel therapeutic modalities are addressed, including MitoSNO and nilotinib, and metalloporphyrins on the basis of the updated paradigm of hepatic I/R injury.

Vascular occlusion or not during liver resection: the continuing story.

BACKGROUND: Vascular occlusion can be applied during liver resection to reduce blood loss. Herein, we provide an update of the current evidence concerning vascular occlusion. METHODS: A systematic literature search was conducted to review the effects of liver in- and outflow occlusion techniques during liver resection, focusing on blood loss and hepatic ischemia-reperfusion injury. RESULTS: The Pringle maneuver (PM) is effective in controlling blood loss; however, there is no indication for routine vascular clamping during hepatic resection in uncomplicated patients. During complex resections and in patients with abnormal liver parenchyma, the intermittent PM is preferred over continuous clamping. Total hepatic vascular exclusion (THVE) is indicated only in resection of tumors involving the inferior caval vein or the caval hepatic junction. THVE can be applied with the preservation of caval vein flow. This mode of selective hepatic vascular exclusion results in less blood loss in combination with the PM. CONCLUSION: If clamping is necessary during complex resections or in abnormal liver parenchyma, intermittent PM is advised. THVE or selective hepatic vascular exclusion may be considered in tumors involving the inferior caval vein or the caval hepatic junction. There is no evidence supporting the use of ischemic preconditioning, maintenance of a low central venous pressure or of pharmacological interventions during liver resection.