Severe liver injury following hepatic ischemia and reperfusion (IR) is an unsolved problem after partial hepatectomy, liver transplantation, traumatic injury of the liver, and after hemorrhagic shock (1). Both local hepatic injury and subsequent systemic organ failure can lead to multiorgan dysfunctions and may contribute to the high mortality of patients with IR injury of the liver (2). Because of the lack of oxygen, hepatocellular injury occurs in the ischemic period triggered by the deficiency of adenosine triphosphate. Despite the fact that tissue oxygen is restored by reperfusion, the hepatocellular injury is aggravated by numerous mechanisms. These are classified as early and late phases of reperfusion injury. With onset of reperfusion, large amounts of reactive oxygen species are released, initiating cell damage (3), e.g., by lipid peroxidation (4). Activation of Kupffer cells leads to the secretion of cytokines, which initiate the late phase of injury. This is characterized by the recruitment of neutrophils and amplification of the inflammatory response with subsequent cell injury (5–7). In the past, several surgical and nonsurgical strategies were developed to attenuate hepatic injury after IR, e.g., pharmacologic and ischemic preconditioning (IPC) (8, 9). Noble gases are one class of agents, which may exert protective conditioning effects in different organs. For example, preconditioning with xenon, argon, neon, and helium attenuates infarction of the myocardium by activating signaling kinases and inhibiting mitochondrial permeability transition (10–12). Furthermore, xenon and helium reduce neuronal injury following hypoxic or ischemic brain injury (13, 14). The unique chemical and physical properties of noble gases could be beneficial in clinical settings, including IR injuries, because the adverse effects of this pharmacologic strategy are negligible (15). In contrast to xenon, helium is characterized by an absence of anesthetic effects. Its use may therefore be indicated for ischemic events where there is no need for general anesthesia. Although cardioprotective effects of helium have been described in experimental and clinical studies, the effects of cardioprotective concentrations of helium on hepatic IR injury have not yet been investigated.
Therefore, it was the aim of our study to evaluate, in a rat model, whether inhalation of helium leads to early preconditioning of warm IR of the liver. A concentration of 70 vol% helium was tested, adopted from previous data that indicated a protective effect of this concentration of helium in ischemic myocardium (10). We hypothesized that helium pretreatment reduces hepatocellular damage after warm IR of the liver. The effect was compared with IPC, known as a strong protective stimulus. In addition, the influence of helium on inflammatory determinants and activation of neutrophils after determining liver IR was ascertained by measuring the serum levels and hepatocellular expression of tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10) and tissue content of myeloperoxidase (MPO). mRNA expression of heme oxygenase 1 (HO-1) was analyzed as one of the most abundant protective proteins in the liver; it has been shown to be induced by preconditioning with volatile anesthetics (16).
MATERIALS AND METHODS
Following approval by the local Animal Use and Care Committee (North Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection, Recklinghausen, Germany), in vivo experiments were performed in accordance with the applicable national guidelines for the care and use of laboratory animals.
Unless stated otherwise, reagents were obtained from Sigma-Aldrich (St Louis, Mo). Pentobarbital was purchased from Rhone-Merieux (Laupheim, Germany); pancuronium was obtained from Inresa (Freiburg, Germany). Pressurized air, medical oxygen, and helium 70 vol% were obtained from Linde Gas (Höllriegelskreuth, Germany). Protease inhibitor was purchased from Roche Diagnostics GmbH (Mannheim, Germany). NP-40 was purchased from The Dow Chemical Company (Midland, Mich). Biotinylated protein ladder was obtained from Cell Signaling Technology Inc (Danvers, Mass). Western blotting luminol reagent was obtained from Santa Cruz Biotechnology Inc (Santa Cruz, Calif). TRIzol Reagent, High Capacity RNA-to-cDNA Master Mix, TaqMan Gene Expression Assay 20× (IL-10/TNF-α/HO-1), TaqMan Gene Expression Assay 20× (GAPDH), and TaqMan Gene Expression Master Mix were obtained from Life Technology Corporation/Invitrogen/Applied Biosystems (Carlsbad, Calif). Antibiotin, horseradish peroxidase–linked antibody was purchased from Cell Signaling Technology Inc. Heme oxygenase 1 (Hsp32) polyclonal antibody was purchased from Enzo Life Science (Plymouth Meeting, Pa). Peroxidase-conjugated AffiniPure donkey anti–rabbit immunoglobulin G and peroxidase-conjugated AffiniPure goat anti–mouse immunoglobulin G were obtained from Jackson ImmunoResearch Laboratories Inc (West Grove, Pa). BD OptEIA rat enzyme-linked immunosorbent assay (ELISA) set for IL-10 and TNF-α were purchased from BD Biosciences (San Diego, Calif).
Animals and in vivo experimental protocol
Male Wistar rats (300 ± 30 g) were anesthetized by an intraperitoneal injection of pentobarbital (60 mg/kg). To ensure an identical metabolic state, the animals were fasted overnight with free access to water. Following tracheal intubation and start of a volume-controlled ventilation with a positive end-expiratory pressure of 3 cmH2O, a central venous access was established, and muscle relaxation was induced with intravenous pancuronium (1 mg/kg) as described previously (16). Administration of pentobarbital to maintain anesthesia (40 mg/kg per hour) and infusion with crystalloid solution (total of 20 mL/kg per hour) were performed continuously. Blood pressure was measured, and blood gas analyzed via a 24-gauge catheter inserted into the carotid artery. Body temperature was measured via a rectal probe and maintained and adapted by heating plates (target temperature 36.5°C–37.5°C). A median laparotomy was performed to expose the portal triad. As described previously, partial hepatic ischemia was induced using a microvascular clip (Aesculap, Tuttlingen, Germany) interrupting the blood flow to the left and middle lobes (70% of the liver). Observation of a pale color discoloration indicated correct positioning of the clip (16). Forty-eight animals were randomized into six groups (n = 8/group): sham: laparotomy without hepatic ischemia; IPC: IPC with 10-min partial ischemia and 10-min reperfusion, HePC: helium preconditioning with three cycles of ventilation with an inhaled gas mixture of helium 70 vol% and oxygen 30 vol% for 5 min, each followed by 5-min washout; IR: 45-min ischemia + 240-min reperfusion; IPC-IR: IPC + IR; HePC-IR: preconditioning with helium + IR. In experimental phases, where helium was not applied and in groups without helium pretreatment, a gas mixture consisting of oxygen 30 vol% and nitrogen 70 vol% was used. To standardize the duration of the experiments to 315 min, the baseline period was extended until the start of the intervention, if necessary (sham, IPC, IR, IPC-IR). At the end of the protocol, animals were killed with an overdose of pentobarbital and exsanguination. Liver tissue was harvested and immediately snap frozen in liquid nitrogen and stored at −70°C. Blood was centrifuged twice for 5 min to separate the serum for subsequent analysis.
The activities of the serum transaminases aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by the standard automated clinical method based on activation of pyridoxal-phosphate and photometric analysis (MODULAR analyzer; Roche Diagnostics). Values are expressed as units per liter.
Quantitative polymerase chain reaction: mRNA expression of IL-10, TNF-α, HO-1
TRIzol-based isolation of total RNA was performed with 100 mg of homogenized liver tissue according to the manufacturer’s instructions. The quantity of RNA was assessed spectrophotometrically (Nanodrop; Thermo Scientific, Wilmington, DE). The integrity and purity of the isolated RNA were verified by agarose gel electrophoresis. High-capacity RNA-to-cDNA Master Mix (Applied Biosystems) was used to obtain cDNA by reverse transcription. The following protocol was applied with a thermocycler (7300 Real-Time PCR System; Applied Biosystems): 25°C for 5 min, 42°C for 30 min, 85°C for 5 min, cooling down to 12°C. TaqMan Gene Expression Master Mix (2X) and TaqMan Gene Expression Assay (20X) (Applied Biosystems) were used for amplification of the cDNA (product IL-10 → Rn00563409; TNF-α → Rn00562055; HO-1 → Rn01536933). Cycler conditions were as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles at 95°C for 25 s, and 60°C for 1 min. For normalization, GAPDH gene was amplified simultaneously (Rn01462661). Samples were run in duplicates. The ΔΔCq method (17) was used to calculate the relative expression of the investigated mRNA with the software tool REST (Relative Expression Software Tool) (18).
To detect the tissue levels of MPO, liver samples were homogenized with 500 µL of 50 mM potassium phosphate buffer, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide. Samples were subjected to three freeze/thaw cycles. After centrifugation, 20 µL of supernatant was mixed with 1.6 mM 3,3′5,5′-tetramethylbenzidine and 3 mM H2O2. After incubation for 5 min, absorbance of the samples was measured spectrophotometrically at 655 nm. Normalization against the content of protein was performed after protein quantification according to Lowry et al. (19).
ELISA of cytokines
Serum levels of TNF-α and IL-10 were determined with a rat-specific ELISA (BD OptEIA at TNF-α/IL-10 ELISA Set; BD Biosciences). The assay was performed according to the manufacturer’s recommendations. Values are reported as picograms per milliliter.
Normal distribution was analyzed throughout all data. Nonparametric data were expressed as median with 25th/75th and 10th/90th percentiles. Normally distributed data were expressed as mean (SD). Comparison between groups was performed by Kruskal-Wallis test (nonparametric) or one-way analysis of variance (normal distribution). Post hoc testing (Dunn or Bonferroni multiple-comparisons test) was performed where appropriate. Level of significance was defined as P < 0.05 (statistical software: GraphPad Prism, version 5.0; GraphPad Software, Inc., La Jolla, Calif). The Relative Expression Software Tool (REST, version 2.0.13) was used to compare the relative expression levels of mRNA (HO-1, IL-10, TNF-α) after pharmacologic or surgical intervention with the values of the sham group. Statistical analyses were approved by an independent statistician.
Acid-base balance, oxygenation, and temperature could have major impact on IR injury of the liver. Therefore, these parameters were measured and recorded (Table 1). Statistical analysis revealed no significant differences between the groups.
Serum levels of AST and ALT
Warm IR of the liver with 45 min of ischemia and 240 min of reperfusion increased AST and ALT serum levels. Ischemic preconditioning as the strongest endogenous protective stimulus greatly reduced the release of these liver enzymes, whereas pretreatment with helium 70 vol% did not alter liver enzyme levels significantly. Neither IPC nor pretreatment with helium 70 vol% without subsequent IR modified liver enzyme release as compared with sham. Data are shown in Figure 1.
mRNA expression of TNF-α and IL-10
To evaluate major proinflammatory and anti-inflammatory mediators, the mRNA expression of TNF-α and IL-10 was measured in liver tissue. Tumor necrosis factor α mRNA expression was increased after IPC, HePC, IR, and HePC-IR compared with sham (Fig. 2A). mRNA expression of IL-10 was increased only after pretreatment with helium 70 vol%. However, pretreatment with helium 70 vol% did not alter the expression of IL-10 after IR (Fig. 2B).
Serum levels of TNF-α and IL-10
The release of TNF-α and IL-10 was measured in the serum to detect the systemic mediator response to protective and inflammatory stimuli. After IR, TNF-α was increased in the serum (P < 0.05 vs. sham), whereas IPC and HePC did not alter IR-induced TNF-α levels significantly (Fig. 3A). The slight increase in IL-10 in the serum after HePC did not reach significance. No differences were detected in the interventional groups as compared with the sham group (Fig. 3B).
Levels of MPO in liver tissue
The level of MPO in liver tissue indicates the degree of degranulation of neutrophils. Reperfusion injury of the liver includes recruitment, accumulation, and degranulation of neutrophils. We analyzed the content of MPO in specimens of ischemic liver. Ischemia and reperfusion induced a significant increase in MPO content, whereas neither IPC-IR nor HePC-IR altered MPO levels (intervention groups vs sham) (Fig. 4A).
mRNA expression of HO-1
Heme oxygenase 1 is part of the most abundant protective intracellular system of the liver, which is—among others—triggered by different agents for pharmacologic preconditioning, e.g., by isoflurane (16). mRNA expression of the primarily transcriptionally regulated HO-1 gene was measured in liver tissue as an early event of its activation. As shown in Figure 4B, IR resulted in a profound increase in the relative expression of HO-1 as compared with sham, IPC, and HePC. Neither IPC nor pretreatment with helium 70 vol% reduced the expression of HO-1 following IR. Whereas IPC alone without subsequent IR increased HO-1 expression as compared with sham, HePC had no effect. In contrast to IPC and IPC + IR, relative expression of HO-1 in HePC-IR was significantly increased compared with HePC.
Ischemia and reperfusion injury of the liver is still a major clinical challenge following partial hepatectomy, liver transplantation, and hemorrhagic shock. In contrast to IPC, no pharmacologic conditioning approach could be introduced into clinical routine so far. The well-described cardioprotective effects of helium make this noble gas an interesting candidate for liver preconditioning, especially in view of its nontoxic and nonanesthetic biophysical properties.
There is evidence that preconditioning with noble gases such as helium, xenon, and argon is able to induce protective effects in cardiac, neuronal, and endothelial tissue. Xenon, a noble gas with anesthetic properties, reduced the infarct size in rat hearts from 50% to 28% after three cycles of preconditioning with 70 vol% xenon (12). The degree of protection was similar to preconditioning with isoflurane and IPC. Pagel et al. (10) extended the hypothesis of cardioprotective noble gases to argon, helium, and neon and confirmed these results for these nonanesthetic agents with the same preconditioning protocol in rabbits. Some studies elucidate the relevance of helium in in vivo experiments with conditions of early preconditioning, late preconditioning, and postconditioning (20, 21). Besides the cardioprotective effects of the noble gases, argon, helium, and xenon were shown to diminish neuronal injury after hypoxia or ischemia in different in vitro and in vivo models (13, 14, 22). Recent data have shown the protective impact of helium preconditioning and postconditioning on human endothelium (23).
So far, only one study has been published that investigated the hepatoprotective effect of helium (24). The aim of this study was to characterize the protective effect of inhalation of 4 vol% hydrogen in a warm liver IR model in mice. Inhalation of 4 vol% helium served as control group. In contrast to hydrogen, inhalation of 4 vol% helium did not show any protective effect on IR injury as assessed by the release of liver enzymes. Huhn et al. (21) demonstrated a concentration-dependent effect of helium in an in vivo model of late preconditioning of the heart. The study indicated a lack of protection with 10 vol% helium, whereas 30 vol%, 50 vol%, and 70 vol% were equally protective. Hence, the concentration of helium used by Fukuda et al. was most likely far below the threshold of any hepatoprotective action.
In the current study, for the first time, we investigated the protective effects of preconditioning with a cardioprotective or neuroprotective concentration of inhaled helium in an in vivo model of warm IR of the liver. No data were available to indicate which protocol for preconditioning with noble gases would be reasonable for hepatic IR. Therefore, the most frequently used protocol for cardiac preconditioning—three cycles of inhalation with 70 vol% of helium for 5-min, each followed by 5 min of washout time—was transferred to our experimental setting. Although evidence exists that lower concentrations of helium applied (30 vol%) induce a comparable degree of protection in the heart at later time points (21), we selected the highest cardioprotective concentration to elucidate a so far unknown effect in our in vivo liver ischemia model. We chose a model of 45 min of partial liver ischemia of the liver followed by 4 h of reperfusion to ensure an adequate increase in liver enzymes as these were expected to reach their peak level between 4 and 6 h after reperfusion (25). Furthermore, this time point marks the end of the early phase of liver IR injury. Animals subjected to IPC with subsequent IR served as positive controls because IPC protects the liver in the setting of IR as described earlier (9). Our results on IPC confirm previous studies and underline the marked reduction in hepatocellular injury after IPC as indicated by a decrease in serum levels of the liver enzymes ALT and AST. These results demonstrate the suitability of the experimental study design to investigate in vivo warm IR injury in rats. In contrast to IPC, we did not detect a protective effect on liver enzyme release after pretreatment with helium.
The early phase of IR injury is characterized by activation of Kupffer cells, which leads to a release of reactive oxygen species and proinflammatory and anti-inflammatory cytokines such as TNF-α and IL-10, respectively (6, 7). We investigated the effect of IPC and helium preconditioning on mRNA expression of TNF-α and IL-10 in the liver and on serum levels of TNF-α and IL-10. Tumor necrosis factor α mRNA expression in the liver was increased as compared with the sham group after each intervention except IPC-IR. The effect of IPC is in line with previous results of other groups (26, 27). In contrast to IPC, HePC did not lead to an attenuated expression in TNF-α mRNA, when followed by IR (HePC-IR). Interestingly, both IPC and HePC abolished the increase in serum levels of TNF-α after IR, suggesting a favorable impact of helium on the release of this major inflammatory cytokine. In contrast to mRNA expression in the liver, serum levels of TNF-α were unaffected by IPC and HePC alone. Although the effect of IR and prior IPC on TNF-α serum levels is well described (25, 28), the impact of inhaled helium after IR was so far unknown and might reflect the protective potential of this noble gas. The lack of a correlation between hepatic mRNA expression and serum levels of TNF-α needs further investigations focusing on different measuring points and different periods of hepatic IR.
Interleukin 10 is involved in endogenous protective mechanisms to diminish hepatic injury after IR of the liver and has been shown to play a significant role in protective effects of IPC. Serafín et al. (29) showed that IL-10 is increased after IPC, and the protective effect of IPC is abolished by an anti–IL-10 antibody. Therefore, we investigated the influence of ischemic and helium preconditioning on mRNA expression and serum levels of IL-10. After helium preconditioning, mRNA expression of IL-10 increased compared with sham. This might contribute to a potential anti-inflammatory effect of helium in the liver. However, the serum levels of IL-10 were not significantly altered by any intervention. This does not necessarily exclude an effect of this interleukin caused by helium preconditioning, because the peak level is reached early with reperfusion and may have declined after 4 h of reperfusion (30).
Tissue MPO has been described to correlate with the number of infiltrated neutrophils and acts as a marker of inflammation (31). We detected a significant increase in MPO activity after IR, which was not observed in other interventional groups compared with sham. Although the increase in MPO activity after hepatic IR is well known (32), the modulation of the inflammatory response after hepatic IR, e.g., infiltration of neutrophils, by helium preconditioning is a new aspect of this pharmacologic intervention. As the neutrophil infiltration of the liver after IR is aggravated particularly in the late phase of reperfusion, the attenuation of the liver injury due to decreased neutrophil infiltration after helium preconditioning is probably not detectable at the early time point of reperfusion investigated in our study.
Heme oxygenase 1 is a cytoprotective protein, mainly induced as part of the oxidative stress response. It catalyzes the degradation of bilirubin to biliverdin, carbon monoxide, and free iron. Whereas biliverdin has antioxidative properties, carbon monoxide mediates anti-inflammatory and antiapoptotic effects (33). Therefore, we investigated the mRNA expression of the primarily transcriptionally regulated HO-1 gene as an early potential mediator of a protective effect. A significant increase in HO-1 mRNA expression was detected after IPC, IR, IPC-IR, and HePC + IR, whereas helium preconditioning alone did not influence the mRNA expression level. Although HO-1 mRNA expression following liver injury represents an endogenous mechanism to ameliorate oxidative stress and to influence vasoregulation (34), the strong increase in HO-1 mRNA expression after IR is also part of the oxidative stress response and correlates with the degree of injury after IR. The increase in HO-1 mRNA after IPC might contribute to the beneficial effect of IPC, and HO-1 is not further induced by subsequent IR. In contrast, HePC did not alter HO-1 mRNA expression. Subsequent IR raised the HO-1 mRNA expression to the same extent as IR without pretreatment, indicating that inhaled helium has no effect on this important protective enzyme system. Ischemia and reperfusion alone increased mRNA expression of HO-1 significantly as compared with the expression level measured after IPC. Thus, the increase in HO-1 gene expression following liver injury may represent an adaptive endogenous mechanism to ameliorate oxidative stress, but HO-1 expression can also be further induced and play a part in the aggravation of injury following a major deleterious impact. This might explain high levels of HO-1 expression after IR and HePC + IR.
In summary, preconditioning with inhaled helium in relevant concentrations modulates the inflammatory response by increasing hepatic IL-10 mRNA expression and decreasing hepatic MPO and serum TNF-α levels after IR. The lack of evidence that these effects of inhaled helium are able to attenuate liver injury after IR needs further investigations with a focus on different time courses of IR.
The authors thank Dr Pablo E. Verde, Coordination Centre for Clinical Studies, University Hospital Duesseldorf, for statistical review and support. The authors also thank native speaker Gil Webster for linguistic review. Furthermore, they also thank Yvonne Grüber and Antje Nebert for excellent technical assistance.
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