Ischemic Liver Lobes Research Articles

Does fasting protect liver from ischemia and reperfusion injury?

To evaluate local and systemic effects of 24-hour fasting in liver ischemia and reperfusion injury. Twenty-one adult male Wistar rats (330-390 g) were submitted to 60 minutes of hepatic ischemia followed by 24 hours of reperfusion. Before the day of the experiment, the animals fasted, but free access to water was allowed. Two groups were constituted: Control: non-fasted, that is, feeding ad libitum before surgical procedure; Fasting: rats underwent previous fasting of 24 hours. Hepatic ischemia was performed using vascular clamp in hepatic pedicle. At 24 hours after liver reperfusion, blood and tissue samples were collected. To analysis, liver lobes submitted to ischemia was identified as ischemic liver and paracaval non-ischemic lobes as non-ischemic liver. We evaluated: malondialdehyde levels, hepatocellular function (alanine aminotransferase, aspartate aminotransferase activities, and both ratio), cytokines (interleukins-6, -10, and tumor necrosis factor-alpha), hepatic ischemia and reperfusion injury (histology). Malondialdehyde measured in non-ischemic and ischemic liver samples, hepatocellular function and cytokines were comparable between groups. Histological findings were distinct in three regions evaluated. Microvesicular steatosis was comparable between 24-hour fasting and non-fasted control groups in periportal region of hepatic lobe. In contrast, steatosis was more pronounced in zones 2 and 3 of ischemic liver samples of fasting compared to control groups. These data indicates that fasting does not protect, but it can be also detrimental to liver submitted to ischemia/reperfusion damage. At that time, using long fasting before liver surgery in the real world may be contraindicated.

Topological approach of liver segmentation based on 3D visualization technology in surgical planning for split liver transplantation.

Split liver transplantation (SLT) is a complex procedure. The left-lateral and right tri-segment splits are the most common surgical approaches and are based on the Couinaud liver segmentation theory. Notably, the liver surface following right tri-segment splits may exhibit different degrees of ischemic changes related to the destruction of the local portal vein blood flow topology. There is currently no consensus on preoperative evaluation and predictive strategy for hepatic segmental necrosis after SLT. To investigate the application of the topological approach in liver segmentation based on 3D visualization technology in the surgical planning of SLT. Clinical data of 10 recipients and 5 donors who underwent SLT at Shenzhen Third People's Hospital from January 2020 to January 2021 were retrospectively analyzed. Before surgery, all the donors were subjected to 3D modeling and evaluation. Based on the 3D-reconstructed models, the liver splitting procedure was simulated using the liver segmentation system described by Couinaud and a blood flow topology liver segmentation (BFTLS) method. In addition, the volume of the liver was also quantified. Statistical indexes mainly included the hepatic vasculature and expected volume of split grafts evaluated by 3D models, the actual liver volume, and the ischemia state of the hepatic segments during the actual surgery. Among the 5 cases of split liver surgery, the liver was split into a left-lateral segment and right tri-segment in 4 cases, while 1 case was split using the left and right half liver splitting. All operations were successfully implemented according to the preoperative plan. According to Couinaud liver segmentation system and BFTLS methods, the volume of the left lateral segment was 359.00 ± 101.57 mL and 367.75 ± 99.73 mL, respectively, while that measured during the actual surgery was 397.50 ± 37.97 mL. The volume of segment IV (the portion of ischemic liver lobes) allocated to the right tri-segment was 136.31 ± 86.10 mL, as determined using the topological approach to liver segmentation. However, during the actual surgical intervention, ischemia of the right tri-segment section was observed in 4 cases, including 1 case of necrosis and bile leakage, with an ischemic liver volume of 238.7 mL. 3D visualization technology can guide the preoperative planning of SLT and improve accuracy during the intervention. The simulated operation based on 3D visualization of blood flow topology may be useful to predict the degree of ischemia in the liver segment and provide a reference for determining whether the ischemic liver tissue should be removed during the surgery.

Melatonin attenuates hepatic ischemia-reperfusion injury in rats by inhibiting NF-κB signaling pathway

BackgroundThe sterile inflammatory response is one of the key mechanisms leading to hepatic ischemia-reperfusion injury. Melatonin has been shown to prevent organ injuries, but its roles in the inflammatory response after hepatic ischemia-reperfusion injury have not been fully explored, especially in late ischemia-reperfusion injury. The present study aimed to investigate the roles and possible mechanisms of melatonin in the inflammatory response after hepatic ischemia-reperfusion injury. MethodsSixty Sprague-Dawley rats were randomly divided into a sham group, ischemia-reperfusion injury group (I/R group), and melatonin-treated group (M + I/R group). The rats in the I/R group were subjected to 70% hepatic ischemia for 45 min, followed by 5 or 24 h of reperfusion. The rats in the M + I/R group were injected with melatonin (10 mg/kg, intravenous injection) 15 min prior to ischemia and immediately before reperfusion. Serum and samples of ischemic liver lobes were harvested for future analysis, and the 7-day survival rate was assessed after hepatic ischemia-reperfusion surgery. ResultsIn comparison with the I/R group, the M + I/R group showed markedly decreased expression levels of inflammatory cytokines (IL-6 and TNF-α) and numbers of apoptotic hepatocytes (P < 0.05). Immunoblotting showed that the expression levels of IL-6, p-NF-κBp65/t-NF-κBp65 and p-IκB-α/t-IκB-α in the M + I/R group were significantly lower than those in the I/R group, and immunofluorescence staining showed that the expression level of p-NF-κBp65 in the M + I/R group was lower than that in the I/R group (P < 0.05). The 7-day survival rates were 20% in the I/R group and 50% in the M + I/R group (P < 0.05). ConclusionsMelatonin downregulated the activity of the NF-κB signaling pathway in the early and late stages of hepatic ischemia-reperfusion injury, alleviated the inflammatory response, protected the liver from ischemia-reperfusion injury, and increased the survival rate.

Intracellular HMGB1 mediating neutrophil extracellular trap formation during liver sterile inflammation

Abstract Neutrophil extracellular traps (NETs) has been recently found exacerbate sterile inflammatory injury during liver ischemia/reperfusion (I/R). High mobility group box-1 (HMGB1) released from damaged cells, mediates liver damage and inflammatory responses after I/R. However, the intracellular role of HMGB1 in NET formation remains unknown. We sought to determine the role of intracellular HMGB1 in NET formation. Myloid cells HMGB1 knockout (LysM-HMGB1 KO) and HMGB1-flox mice were subjected to a non-lethal warm liver I/R. Less NETs was identified in ischemic liver lobes of LysM-HMGB1 KO mice compared with flox mice by confocal immunofluorescence (IF) imaging. This was associated with significantly less NET markers in LysM-HMGB1 KO mice, serum level of myeloperoxidase (MPO)-DNA complexes, and tissue level of citrullinated histone H3. Absence of HMGB1 in myloid cells protected the live from I/R injury indicated by significantly decreased serum transaminases (sALT), less necrosis and inflammatory cytokines production compared to flox mice. In vitro, no NET formation was observed in HMGB1 KO neutrophils under stimulation of PMA (positive control) or extracellular HMGB1 or histones confirmed by IF imaging. Chromatin in HMGB1 KO neutrophils failed to de-condensate in response to PMA which is the key step of NET formation. Additionally, gene deletion of HMGB1 in neutrophils completely blocked citrullination of histone H3 as a key step of NET formation, compared with control neutrophils in response to various stimulations. Our study demonstrates the dominant role of intracellular HMGB1 in NET formation. Lack of HMGB1 in neutrophils leads to diminished NET formation protect the liver from organ damage and cells death after liver I/R.

The relationship between activation of Toll-like receptor 4 and partial hepatic ischemia/reperfusion injury in mice

Objective To investigate the relationship between the activation of Toll-like receptor 4 (TLR4) and hepatic ischemia reperfusion injury. Methods 24 mice were randomly divided into TLR defect group (C3H/Hej, Hej group), wild type group (/Heouj C3H, Heouj group) and sham operation group (Sham group), 8 mice in every group. BALB/c mice copied partial hepatic ischemia reperfusion injury model, using immune histochemistry method to observe the TLR4 in the distribution of liver and the expression while monitoring of plasma alanine amino transferase (ALT) and portal vein endotoxin in blood serum endotoxin (EN) and tumor necrosis factor-α (TNF-α) changes; then to TLR4 congenital defects in mice C3H/Hej and wild type mice C3H/Heouj model and observe changes of ALT and portal vein serum TNF-α. Results (1) BALB/c mouse the left middle lobe of liver ischemia after 1 h, reperfusion 1, 3 h, ischemic liver lobe TLR4 increased expression of and 1 h after reperfusion, the strongest, positive cells is mainly Kupffer cells and blood vessels in neutrophils. (2) There was no significant difference between the time points of portal vein serum EN (P>0.05). (3) At 3 h of repefusion, serum TNF-α was significantly higher than sham group [Hej: (152±43) pg/ml vs. (18±10) pg/ml, n=6, t=5.26, P<0.01; Heouj: (249±52) pg/ml vs. (25±13) pg/ml, n=6, t=7. 24, P<0.01]; (4) Hej mice liver function damage light in Heouj mice [reperfusion for 1 h: (662±106) vs. (1 216±174) pg/ml, n=6, t=4.21, P<0.01; reperfusion for 3 h: (1145±132) pg/ml vs. (2 958±187) pg/ml, n=6, t=13.72, P<0.01], and 3 h of reperfusion the level of serum TNF-α significantly lower than heouj mice [(152±43) pg/ml vs. (249±52) pg/ml, n=6, t=3.94, P<0.01]. Conclusion TLR4 receptor is activated during hepatic ischemia reperfusion injury, the process of hepatic ischemia reperfusion injury was mediated by TNF-α and other cytokines. Key words: Liver; Reperfusion, injury; Receptors; Endotoxin

New NIR spectroscopy based method to determine ischemia in vivo in liver – a first study on rats

Abstract In this study we analysed ischemic rat liver lobes with Near Infrared Spectroscopy (NIRS) to evaluate the potential of NIRS as predictor of ischemic reperfusion injury (IRI). We were able to predict duration of ischemia using visible and near infrared light.

Damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury.

Innate immunity plays a crucial role in the response to sterile inflammation such as liver ischemia/reperfusion (I/R) injury. The initiation of liver I/R injury results in the release of damage-associated molecular patterns, which trigger an innate immune and inflammatory cascade through pattern recognition receptors. Neutrophils are recruited to the liver after I/R and contribute to organ damage and innate immune and inflammatory responses. Formation of neutrophil extracellular traps (NETs) has been recently found in response to various stimuli. However, the role of NETs during liver I/R injury remains unknown. We show that NETs form in the sinusoids of ischemic liver lobes in vivo. This was associated with increased NET markers, serum level of myeloperoxidase-DNA complexes, and tissue level of citrullinated-histone H3 compared to control mice. Treatment with peptidyl-arginine-deiminase 4 inhibitor or DNase I significantly protected hepatocytes and reduced inflammation after liver I/R as evidenced by inhibition of NET formation, indicating the pathophysiological role of NETs in liver I/R injury. In vitro, NETs increase hepatocyte death and induce Kupffer cells to release proinflammatory cytokines. Damage-associated molecular patterns, such as High Mobility Group Box 1 and histones, released by injured hepatocytes stimulate NET formation through Toll-like receptor (TLR4)- and TLR9-MyD88 signaling pathways. After neutrophil depletion in mice, the adoptive transfer of TLR4 knockout or TLR9 knockout neutrophils confers significant protection from liver I/R injury with a significant decrease in NET formation. In addition, we found inhibition of NET formation by the peptidyl-arginine-deiminase 4 inhibitor and that DNase I reduces High Mobility Group Box 1 and histone-mediated liver I/R injury. Damage-associated molecular patterns released during liver I/R promote NET formation through the TLR signaling pathway. Development of NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R.

Neutrophil extracellular traps activated by damage associated molecular patterns exacerbates sterile inflammatory liver injury (INM1P.427)

Abstract The initiation of liver ischemia/reperfusion (I/R) injury results in the release of damage associated molecular patterns (DAMPs) such as HMGB1 and histones, which trigger innate immune and inflammatory cascade via Toll-like receptor 4 (TLR4) or TLR9. Infiltrated neutrophils contribute to the organ damage, innate immune and inflammatory responses after liver I/R. Formation of neutrophil extracellular trap (NET) has been recently found in response to various stimuli. However, the role of NETs during liver I/R remains unknown. We show that NETs form in the sinusoids of ischemic liver lobes in vivo, associated with increased serum level of myeloperoxidase (MPO)-DNA complexes and tissue level of citrullinated-histone H3 (NET markers) compared to control mice. Treatment with peptidyl-arginine-deiminase (PAD) 4 inhibitor or DNase I conferred significant protection after liver I/R evidenced by inhibition of NET formation, indicating the pathophysiological role of NETs in liver I/R injury. DAMPs, such as HMGB1 and histones stimulate NET formation through TLR4 and TLR9-MyD88 signaling pathways. After neutrophil depletion in mice, the adoptive transfer of TLR4 knockout (KO) or TLR9 KO neutrophils confers significant protection from liver I/R injury with significant decrease in NET formation. Conclusion: DAMPs released during liver I/R promotes NET formation through TLRs signaling pathway. NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R.

CXC chemokines function as a rheostat for hepatocyte proliferation and liver regeneration.

BackgroundOur previous in vitro studies have demonstrated dose-dependent effects of CXCR2 ligands on hepatocyte cell death and proliferation. In the current study, we sought to determine if CXCR2 ligand concentration is responsible for the divergent effects of these mediators on liver regeneration after ischemia/reperfusion injury and partial hepatectomy.MethodsMurine models of partial ischemia/reperfusion injury and hepatectomy were used to study the effect of CXCR2 ligands on liver regeneration.ResultsWe found that hepatic expression of the CXCR2 ligands, macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), was significantly increased after both I/R injury and partial hepatectomy. However, expression of these ligands after I/R injury was 30-100-fold greater than after hepatectomy. Interestingly, the same pattern of expression was found in ischemic versus non-ischemic liver lobes following I/R injury with expression significantly greater in the ischemic liver lobes. In both systems, lower ligand expression was associated with increased hepatocyte proliferation and liver regeneration in a CXCR2-dependent fashion. To confirm that these effects were related to ligand concentration, we administered exogenous MIP-2 and KC to mice undergoing partial hepatectomy. Mice received a “high” dose that replicated serum levels found after I/R injury and a “low” dose that was similar to that found after hepatectomy. Mice receiving the “high” dose had reduced levels of hepatocyte proliferation and regeneration whereas the “low” dose promoted hepatocyte proliferation and regeneration.ConclusionsTogether, these data demonstrate that concentrations of CXC chemokines regulate the hepatic proliferative response and subsequent liver regeneration.

RhoA/ROCK Signaling Triggers Innate Immune Responses in Ischemia-Stressed Liver Through Activation of c-Jun N-Terminal Kinase Pathway.

Background: The RhoA/Rho kinase (ROCK) signaling plays an important role in innate immune cell activation and the development of inflammatory diseases. However, little is known on the functional role of RhoA/ROCK signaling in the activation of innate immune responses during organ ischemia-reperfusion injury (IRI). This study was designed to explore potential molecular mechanisms of Rho/ROCK signaling in innate immune responses triggered by IR stress in a mouse liver. Methods: Using a partial hepatic warm ischemia model (90 min), C57BL/6 wide-type (WT) mice (n=6/gr) were pretreated with ROCK inhibitor H1152 (3 mg/kg, i.p.) or vehicle at 1h prior to ischemia, and then sacrificed at 6 h of reperfusion. In parallel in vitro study, bone marrow-derived WT macrophages (BMMs) were transfected with JNK1 siRNA/nonspecific siRNA (100 nM) or treated with H1152 (10μM), and then incubated with LPS (100 ng/ml). Results: Hepatic IR increased ROCK1 and c-Jun N-terminal kinase (JNK) expression and enhanced PTEN activity, whereas anti-ROCK (H1152) treatment decreased JNK and PTEN/TLR4 activation yet increased PI3K/Akt expression. Unlike in vehicle-treated WT controls, H1152 preconditioning improved hepatic function, reduced hepatocellular damage, and depressed macrophage/neutrophil trafficking, pro-inflammatory cytokine programs, and hepatocellular necrosis/apoptosis, while increasing anti-apoptotic functions in ischemic liver lobes. LPS-stimulated BMMs in vitro revealed increased expression of ROCK1, JNK, PTEN, and TLR4. However, knockdown of JNK with siRNA readily suppressed PTEN/TLR4, leading to diminished NF-κB activation and decreased expression of proinflammatory mediators. Consistent with our in vivo findings, pretreatment of LPS-stimulated BMMs with H1152 decreased JNK expression and upregulated Akt phosphorylation. Conclusion: This study demonstrates that IR-triggered RhoA/ROCK signaling induced JNK activation, leading to enhanced PTEN/TLR4 signaling. Inhibition of RhoA/ROCK protected livers from IR-mediated inflammation. Knockdown of JNK depressed PTEN yet promoted PI3K/Akt, which in turn negatively regulated TLR4 activation. By documenting the critical role of RhoA/ROCK-JNK signaling cascade in the mechanism of innate immune-driven local inflammation responses, our findings provide the rationale for a novel therapeutic strategy against IRI in organ transplant recipients.

Immunoglobulin M, C-Reactive Protein and Complement Activation in Rat Hepatic Ischemia-Reperfusion Injury

Background: Ischemia-reperfusion (I/R) models have shown that C-reactive protein (CRP) and immunoglobulin M (IgM) are involved in complement activation. Binding of CRP and IgM to damaged cell membranes initiates complement activation and aggravates I/R injury in various organs. However, the time course of CRP- and IgM-mediated complement activation and the relation to hepatocellular injury and inflammation in liver I/R are unknown. Aim: To evaluate the time course of IgM- and CRP-related complement activation and the relation to hepatocellular injury and inflammation in a hepatic I/R rat model. Methods: Male Wistar rats were allocated to (1) five groups of animals exposed to 60 min of partial ischemia (70%) induced via clamping of the left segmental portal triad, followed by 0, 3, 6, 12 or 24 h of reperfusion (n = 6 in each group); (2) five groups of sham-operated animals with corresponding reperfusion times (n = 5), and (3) a control group sacrificed before ischemia (n = 5). Hepatocellular injury, inflammatory response, rat plasma CRP and IgM levels and immunohistochemical depositions of CRP, IgM and C3 were assessed for each group. Results: Histopathological injury scores of hematoxylin and eosin sections of ischemic liver lobes demonstrated increasing values throughout the reperfusion time with a peak at 12 h. Plasma aminotransferases (alanine aminotransferase and aspartate aminotransferase) significantly increased after 3 h of reperfusion, peaking at 6 h (3,100 ± 800 U/l; p < 0.05). Hepatic neutrophil influx significantly increased from 3 to 6 h of reperfusion (p < 0.05) and demonstrated the highest value at 12 h (1.1 ± 0.2 U/mg of protein). Plasma IL-6 levels in the ischemia groups showed peak values after 6 h of reperfusion, decreasing significantly thereafter (p < 0.05). Plasma CRP values reached highest levels after 3 h of reperfusion (mean 91 ± 5% of control pool), decreasing significantly thereafter. Rat IgM concentrations in plasma did not significantly change throughout the reperfusion time. Immunohistochemical depositions of IgM, CRP and C3 in ischemic lobes demonstrated a similar pattern in time, reaching maximum values at 12 h of reperfusion. The percentages of depositions of CRP and IgM were significantly correlated [r(S) = 0.569; p < 0.001; Spearman test]. The time course of C3 and CRP depositions throughout reperfusion and C3 and IgM staining were significantly similar [r(S) = 0.797 and r(S) = 0.656, respectively; p < 0.0001; ANOVA]. Conclusions: CRP and IgM depositions demonstrate a parallel time course throughout the reperfusion to hepatocellular damage, inflammatory response and activated complement deposition in this rat hepatic I/R model. Furthermore, the time course of CRP and IgM depositions was significantly similar to that of activated complement depositions.

90 CYCLOSPORINE AND TACROLIMUS HAVE DIFFERENT INHIBITORY EFFECTS ON TOLL-LIKE RECEPTOR SIGNALLING IN HEPATITIS C RECURRENCE POST LIVER TRANSPLANTATION

Background and Aims: Ischemia-reperfusion injury (IRI) is a key factor that contributes to early and late dysfunction of liver grafts. Recent studies reveal that natural killer (NK) cells play an important role in liver injury post IRI. We hypothesized that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand with high expression on NK cells significantly impacts IRI. Methods: C57/BL6 wild-type and TRAIL knock-out mice (TRAIL−/−) were subjected to hepatic IRI for one hour. Adoptive transfer of wild-type and TRAIL−/− NK cells was performed into RAG2/common gamma null mice that lack T, B and NK cells. Liver injury was assessed by hepatic neutrophil infiltration, alanine aminotransferase (ALT), aspartate transaminase (AST), hepatic neutrophil activation by myeloperoxidase (MPO) activity. NK cell subsets of the ischemic liver lobe were analysed by flow cytometry. NK cell cytotoxicity and interferon gamma secretion were performed in vitro studies. Results: TRAIL−/− mice exhibit significantly more hepatic damage assessed by AST and ALT levels 24 hours post IRI compared to wildtype mice. Adoptive transfer of TRAIL−/− NK cells to Rag2/common gamma-null mice was associated with significantly increased IRI compared to transfer with wild-type NK cells. Hepatic neutrophil activation was significantly increased in TRAIL−/− compared to wild-type mice. Staining for CD107a, a marker of degranulation and cytotoxicity on NK cells was significantly elevated in ischemic liver lobes of TRAIL−/− compared to wild-type mice. In vitro NK cell cytotoxicity to a Yac-1 cancer cell line was significantly increased in sorted TRAIL−/− NK cells compared to wild-type NK cells. Systemic cytokine levels (TNF alpha, IL-1beta, IL-6) and interferon gamma secretion of NK cells after stimulation with IL-12/IL-18 in vitro were not significantly different between the groups. Conclusions: These results show that expression of TRAIL on NK cells is protective in a murine model of hepatic IRI via the modulation of NK cell mediated cytotoxicity.

89 DELETION OF TRAIL ON NK CELLS IS ASSOCIATED WITH EXCESSIVE HEPATIC ISCHEMIA-REPERFUSION INJURY IN MICE

Background and Aims: Ischemia-reperfusion injury (IRI) is a key factor that contributes to early and late dysfunction of liver grafts. Recent studies reveal that natural killer (NK) cells play an important role in liver injury post IRI. We hypothesized that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand with high expression on NK cells significantly impacts IRI. Methods: C57/BL6 wild-type and TRAIL knock-out mice (TRAIL−/−) were subjected to hepatic IRI for one hour. Adoptive transfer of wild-type and TRAIL−/− NK cells was performed into RAG2/common gamma null mice that lack T, B and NK cells. Liver injury was assessed by hepatic neutrophil infiltration, alanine aminotransferase (ALT), aspartate transaminase (AST), hepatic neutrophil activation by myeloperoxidase (MPO) activity. NK cell subsets of the ischemic liver lobe were analysed by flow cytometry. NK cell cytotoxicity and interferon gamma secretion were performed in vitro studies. Results: TRAIL−/− mice exhibit significantly more hepatic damage assessed by AST and ALT levels 24 hours post IRI compared to wildtype mice. Adoptive transfer of TRAIL−/− NK cells to Rag2/common gamma-null mice was associated with significantly increased IRI compared to transfer with wild-type NK cells. Hepatic neutrophil activation was significantly increased in TRAIL−/− compared to wild-type mice. Staining for CD107a, a marker of degranulation and cytotoxicity on NK cells was significantly elevated in ischemic liver lobes of TRAIL−/− compared to wild-type mice. In vitro NK cell cytotoxicity to a Yac-1 cancer cell line was significantly increased in sorted TRAIL−/− NK cells compared to wild-type NK cells. Systemic cytokine levels (TNF alpha, IL-1beta, IL-6) and interferon gamma secretion of NK cells after stimulation with IL-12/IL-18 in vitro were not significantly different between the groups. Conclusions: These results show that expression of TRAIL on NK cells is protective in a murine model of hepatic IRI via the modulation of NK cell mediated cytotoxicity.

Induction of Different Types of Cell Death After Normothermic Liver Ischemia-Reperfusion

Normothermic liver ischemia-reperfusion (I-R) may induce hepatocellular autophagy, apoptosis, and necrosis. The aim of this study was to investigate these three types of cell death in normothermic liver I-R in rats. A segmental normothermic ischemia of the liver was induced for 120 minutes. Liver autophagy was evaluated by transmission electron microscopy and LC3 (Light Chain 3) immunohistochemical studies. Liver apoptosis was assessed by FLIVO (FLuorescence in vIVO) and TUNEL (TdT-mediated dUTP nick end labeling) assays. Liver necrosis was determined by optical microscopic examination. Autophagy was increased in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes. Fluorescence microscopy showed in situ caspase-3 and -7 specific activity to be increased in ischemic liver lobes after 6 hours of reperfusion, compared with nonischemic lobes. Quantitative analysis of apoptotic cells evaluated by the TUNEL method showed a clearly significant increase in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes. Necrotic cell death was significantly increased in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes ( P < .005). In conclusion, 120 minutes normothermic liver I-R resulted in increased autophagic, apoptotic and necrotic cell death.

Liver Apoptosis Following Normothermic Ischemia-Reperfusion: In Vivo Evaluation of Caspase Activity by FLIVO Assay in Rats

Normothermic liver ischemia-reperfusion (I-R) may induce hepatocellular apoptosis. Caspase activation is involved in the initiation and execution of apoptosis. The aim of this study was to determine in vivo caspase activity in normothermic liver I-R in rats. Segmental normothermic ischemia of the liver was induced for 120 minutes in rats. After intravenous injection of the green probe FLIVO ™, in vivo caspase-3- and -7-specific activity was determined using fluorescence microscopy, in either nonischemic or ischemic liver lobes at 3 and 6 hours after reperfusion. Liver apoptosis was assessed by the deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Fluorescence microscopy showed that in vivo caspase-3- and -7-specific activities were significantly increased ( P < .005) in ischemic lobes at 3 and 6 hours of reperfusion, compared with nonischemic liver lobes. Quantitative analysis of apoptotic cells measured by the TUNEL method showed a significant increase among apoptotic cells in ischemic lobes at 3 and 6 hours after reperfusion ( P < .005), compared with nonischemic liver lobes. In conclusion, 120-minute normothermic liver I-R resulted in increased caspase-3- and -7-specific activities and in liver cell apoptosis.

JunD Protects the Liver from Ischemia/Reperfusion Injury by Dampening AP-1 Transcriptional Activation

The AP-1 transcription factor modulates a wide range of cellular processes, including cellular proliferation, programmed cell death, and survival. JunD is a major component of the AP-1 complex following liver ischemia/reperfusion (I/R) injury; however, its precise function in this setting remains unclear. We investigated the functional significance of JunD in regulating AP-1 transcription following partial lobar I/R injury to the liver, as well as the downstream consequences for hepatocellular remodeling. Our findings demonstrate that JunD plays a protective role, reducing I/R injury to the liver by suppressing acute transcriptional activation of AP-1. In the absence of JunD, c-Jun phosphorylation and AP-1 activation in response to I/R injury were elevated, and this correlated with increased caspase activation, injury, and alterations in hepatocyte proliferation. The expression of dominant negative JNK1 inhibited c-Jun phosphorylation, AP-1 activation, and hepatic injury following I/R in JunD-/- mice but, paradoxically, led to an enhancement of AP-1 activation and liver injury in JunD+/- littermates. Enhanced JunD/JNK1-dependent liver injury correlated with the acute induction of diphenylene iodonium-sensitive NADPH-dependent superoxide production by the liver following I/R. In this context, dominant negative JNK1 expression elevated both Nox2 and Nox4 mRNA levels in the liver in a JunD-dependent manner. These findings suggest that JunD counterbalances JNK1 activation and the downstream redox-dependent hepatic injury that results from I/R, and may do so by regulating NADPH oxidases.

Ischemic preconditioning enhances regenerative capacity of hepatocytes in long‐term ischemically damaged rat livers

Ischemic preconditioning (IPC) protects tissues against ischemia and reperfusion (I/R) injury. The aim of this study was to examine the impact of IPC on protection and regeneration of hepatocytes after prolonged I/R injury. A rat model of segmental (70%) hepatic ischemia was used to determine the effect of 10-min IPC preceding 40, 60, 90, or 120 min of liver ischemia. The effect was assessed by comparing cytolysis markers and necrotic areas of the liver, as well as the regenerative capacity of hepatocytes using the proliferating cell nuclear antigen labeling index (PCNA-LI) and weight of the ischemic liver lobe. Protein kinase B/Akt (Akt) and caspase-9 were investigated immunohistochemically to determine the effect of IPC on activation of survival and anti-apoptotic signals. In the model of 40 min I/R, which resulted in focal necrosis of the liver, IPC significantly protected against I/R injury by reducing the area of focal necrosis, level of PCNA-LI and immunoreactivities to Akt and caspase-9. In contrast, IPC did not prevent ischemic damage in the 90- and 120-min ischemic model with massive liver necrosis. However, IPC enhanced the regenerative capacity of the remaining hepatocytes with higher levels of PCNA-LI, number of Akt-positive cells and mean weight of the liver lobe postoperatively than in the controls. In a model of focal necrosis of the liver, IPC protected hepatocytes against I/R injury. In addition, in a model of massive necrosis, IPC maintained the regenerative capacity of the remaining hepatocytes by enhancing the survival signals.

The Effect of Ischemic Preconditioning Prior to Intraoperative Radiotherapy on Ischemic and on Reperfused Rat Liver

The purpose of this study was to increase the tolerance of the liver to radiation injury with the proven effect of ischemic precondition (IP) in decreasing oxygen-derived free radicals, and to compare the effect of intraoperative radiotherapy (IORT) during ischemia and during reperfusion on rat liver. Two hundred fifty to 280 g male Wistar rats underwent 45 min of normothermic, segmental liver ischemia with or without IP/5 min ischemia and 10 min reperfusion, in two cycles. During ischemia or reperfusion, IORT doses of 0, 25, or 50 Gy were applied to the ischemic liver lobe. Hepatic microcirculation was monitored by laser Doppler flowmeter. Short- and long-term histological, alkaline phosphatase, bilirubin and tumor necrosis factor-alpha levels, liver tissue, and serum antioxidant alterations were measured. Histological, laboratory, as well as flowmetry alterations caused by 25 Gy were reversible after 6 mo. Three mo following IORT, histological examination revealed parenchymal fibrosis, bridging, liver cell atrophy, and bile duct proliferation in the group that was irradiated with 50 Gy during reperfusion, without IP. In this group, the changes were present 6 mo following IORT, and also the levels of tumor necrosis factor-alpha and oxygen-derived free radicals after reperfusion were increased. All these changes were significantly milder in groups with IP, especially those that were irradiated during ischemia. IORT to the liver, up to 25 Gy, can be applied without short- or long-term treatment morbidity. Doses of up to 50 Gy are tolerated with IP, which has never been described before. Irradiation during ischemia is less toxic for the liver tissue.

Endogenous danger signals trigger hepatic ischemia/reperfusion injury through toll-like receptor 4/nuclear factor-kappa B pathway

Restoration of blood flow to the ischemic liver lobes may paradoxically exacerbate tissue injury, which is called hepatic ischemia/reperfusion injury (IRI). Toll-like receptor 4 (TLR4), expressed on several liver cell types, and the nuclear factor-kappa B (NF-kappaB) signaling pathway are crucial to mediating hepatic inflammatory response. Because IRI is essentially a kind of profound acute inflammatory reaction evoked by many kinds of danger signals, we investigated TLR4/NF-kappaB signaling pathway activation in a murine model of partial hepatic IRI. Wild-type mice (WT, C3H/HeN) or TLR4 mutant mice (C3H/HeJ) were subjected to 45 minutes of partial hepatic ischemia followed by 1 hour, 3 hours of reperfusion. Sham group accepted the same procedure without the obstruction of blood supply. At the end of reperfusion, the compromise of liver function and the histological change of liver sections were measured as the severity of liver injury. The level of endotoxin in the portal vein was measured by limulus assay. NF-kappaB activation was determined by electrophoretic mobility shift assay (EMSA). The levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in systemic blood after hepatic IRI were assessed by enzyme-linked immunosorbent assay (ELISA). The compromise of liver function and the morphological injuries in mutant mice were relieved more markedly than those in WT mice after partial hepatic IRI. NF-kappaB activation in WT mice was stronger than that in TLR4 mutant mice, and both were stronger than those in the sham operated mice (P < 0.01). Endotoxin in each group was undetectable. The levels of TNF-alpha and IL-1beta in systemic blood were elevated in both strains, but lower in the sham operated group. These mediators were significantly decreased in TLR4 mutant mice compared with those in WT mice (P < 0.01). The TLR4/NF-kappaB signaling pathway may mediate hepatic IRI triggered by endogenous danger signals. Inhibition of the TLR4/NF-kappaB pathway may be a potential therapeutic target for attenuating ischemia/reperfusion-induced tissue damage in some clinical settings.

Alterations in Protein Tyrosine Kinase Pathways in Rat Liver Following Normothermic Ischemia-Reperfusion

The phosphoregulation of signal transduction pathways is a complex series of reactions that modulate the cellular response to ischemia-reperfusion (I-R). The aim of this study was to evaluate the effect of normothermic liver I-R on protein tyrosine phosphorylation, production of angiogenic growth factors, and activation of signal proteins in tyrosine kinase pathways. A segmental normothermic ischemia of the liver was induced in rats by occluding the blood vessels (including the bile duct) to the median and left lateral lobes for 120 minutes. Liver extracts from either ischemic or nonischemic lobes were prepared at 0, 1, 3, and 6 hours after reperfusion. Liver tyrosine phosphorylation of proteins was examined by Western blot analysis, whereas vascular endothelial growth factor (VEGF) mRNA was analyzed by Northern blot. In ischemic liver lobes, VEGF mRNA and total protein levels increased at 1 and 3 hours after reperfusion. Tyrosine phosphorylation of the VEGF receptor Flk-1 and the platelet-derived growth factor receptor (PDGF-R) was increased only at 1 hour after reperfusion, while c-Src tyrosine phosphorylation remained increased at 3 hours and remained up to 6 hours after reperfusion. In conclusion, 1-R led to alterations in protein tyrosine phosphorylation and increased expression of VEGF in rat liver.