Ischemic Lobes Research Articles

Role of vascular cell adhesion molecule-1 in the mouse model of hepatic ischemia/reperfusion and the hematogenic metastasis

To investigate the effect of ischemia/reperfusion (I/R) on tumor metastasis in a experimental mouse model of hematogenous metastasis after I/R and to quantify expression of vascular cell adhesion molecule-1 (VCAM-1) during I/R. An experimental mouse model of metastasis after partial hepatic I/R was designed to determine the effects of I/R on tumor metastasis to liver. Tumor loads were valued 14 days after operation. In addition, the expressions of alanine transaminase (ALT), aspartate transaminase (AST), and VCAM-1 were detected. Two hours after hepatic reperfusion, ALT and AST levels in ischemia 45-minute group and ischemia 30-minute group were significantly higher than in the sham group (all P < 0.05). Also, the changes of ALT and AST were more obvious in the ischemia 45-minute group than in ischemia 30-minute group (all P < 0.05). In the sham group, both ALT and AST slightly and transiently increased. ALT and AST in the ischemia 45-minute group and ischemia 30-minute group at 8 hours were both significantly higher than those at 2 hours reperfusion (P<0.05). The tumor load (valued by hepatic replacement area) and the expression of VCAM-1 in ischemic lobe were significantly larger in the ischemia 45-minute group than in the ischemia 30-minute group and sham group (P = 0.013, P = 0.007). However, there was no statistical difference on tumor load between the right lobe of sham operated mice and the right lobe (nonischemic lobes) of mice subjected to I/R (P = 0.089). Mouse survivals were significantly longer in the sham group than in the ischemia 30-minute group (P = 0.041) but were not significantly different between the ischemia 45-minute group and ischemia 30-minute group (P = 0.055). VCAM-1 expression in ischemia 45-minute group was significantly higher than in ischemia 30-minute group and sham group(P = 0.003, P < 0.001), and it was positively correlated with the hepatic replacement area (r = 0.491, P = 0.045). Hepatic I/R promotes liver hematogenic metastasis of hepatocellular carcinoma in mice and at least in part, through the induction of VCAM-1 expression.

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.

Anton's Syndrome due to Bilateral Ischemic Occipital Lobe Strokes.

We present a case of a patient with Anton's syndrome (i.e., visual anosognosia with confabulations), who developed bilateral occipital lobe infarct. Bilateral occipital brain damage results in blindness, and patients start to confabulate to fill in the missing sensory input. In addition, the patient occasionally becomes agitated and talks to himself, which indicates that, besides Anton's syndrome, he might have had Charles Bonnet syndrome, characterized by both visual loss and hallucinations. Anton syndrome, is not so frequent condition and is most commonly caused by ischemic stroke. In this particular case, the patient had successive bilateral occipital ischemia as a result of massive stenoses of head and neck arteries.

Protective effects of α-lipoic acid and L-carnitine in liver ischemia/reperfusion injury.

Objective: Oxidative stress plays an important role in the pathogenesis of liver ischemia/ reperfusion injury. Thus, antioxidant treatment can be protective against to liver ischemia/reperfusion injury. The aim of this study to investigate the effects of α-lipoic acid and L-carnitine on liver total oxidant status, lipid peroxidation, protein oxidation, neutrophil infiltration and hepatic necrosis in liver ischemia/reperfusion model. Methods: Wistar albino male rats were divided into four groups randomly: Sham (n=7), ischemia/reperfusion (n=7), α-lipoic acid (n=8) and L-carnitine (n=8). α-Lipoic acid (100 mg/kg) and L-carnitine (100 mg/kg) were given intraperitoneally to α-lipoic acid group 15 minutes before and to L-carnitine group 30 minutes before ischemia/reperfusion protocol, respectively. To induce hepatic ischemia/reperfusion injury, ischemia (60 minutes) and reperfusion (30 minutes) were applied to all groups except sham group. Total oxidant status, malondialdehyde, advanced oxidation protein products and myeloperoxidase levels were measured in ischemic lobes of liver tissues. Hepatic necrosis was scored microscopically. Results: There was no significant change in myeloperoxidase levels as an indicator of neutrophil infiltration after reperfusion procedure. Both L-carnitine and α-lipoic acid caused a significant decrease in hepatic necrosis. While L-carnitine prevents an increase in total oxidant status, lipid peroxidation and protein oxidation, α-lipoic acid prevents only an increase in lipid peroxidation of the liver in hepatic ischemia/reperfusion injury. Conclusion: As a result; we can report that L-carnitine and α-lipoic acid have protective effects against to hepatic ischemia/reperfusion injury.

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.

IL-17A-producing NK cells were implicated in liver injury induced by ischemia and reperfusion

NK cells play a critical role in several types of liver injury. The aim of this study was to evaluate the role of NK cells in liver ischemia reperfusion injury (IRI) and the underlying mechanism.Male Rag1−/− mice and wild type mice were subjected to partial hepatic IRI. Anti-NK1.1 (300μg/mouse, ip) was used to deplete NK cells. Liver injury was evaluated by level of serum alanine aminotransferase (ALT). Hepatic inflammatory cytokines, neutrophils and CXCL-2 expression were measured following ischemia and reperfusion. Additionally, NK cells were cultured with or without IL-6, IL-21, IL-23 and IL-10 for 24h, then IL-17A level in the supernatants was analyzed by ELISA. Production of IL-17A was increased in NK cells after reperfusion. Various cytokines such as, IL-6, IL-21 and IL-23, which also elevated after IRI, can promote IL-17A production and up-regulate the phosphorylation of STAT3 in NK cells, while the increase was repressed in the presence of IL-10. Depletion of NK cells decreased IL-17A level in Rag1−/− mice ischemic lobes. Meanwhile, hepatic infiltration of neutrophils and CXCL-2 level were reduced and liver injury was ameliorated. Neutralization of IL-17A was used to confirm the role of this cytokine produced by NK cells in Rag1−/− mice. In conclusion, at initial stage of liver IRI, NK cells increase IL-17A production and promote liver injury.

Role of UCP2 Expression after Hepatic Warm Ischemia-Reperfusion in the Rat

Background/AimsThe role of uncoupling protein-2 (UCP2) in the liver is currently unclear. Emerging evidence suggests a relationship between UCP2 and oxidative stress. In the present study, we tested the hypothesis that UCP2 expression in the liver might change during warm ischemia-reperfusion (I/R) according to oxidative stress.MethodsWistar rats were subjected to 40 (short ischemia) or 90 (long ischemia) minutes of partial lobar ischemia followed by 4 hours of reperfusion. UCP2 expression in the ischemic and nonischemic lobes was assessed using reverse transcription-polymerase chain reaction and immunohistochemistry. Malondialdehyde concentrations in the liver tissue were also compared.ResultsMalondialdehyde concentrations in the ischemic lobes were significantly higher in the long ischemia group. In the ischemic lobes of the short ischemia group, UCP2 protein expression was induced in hepatocytes, which did not express the protein prior to treatment, and the expression levels were higher than in the long ischemia group. The intralobular distribution of UCP2 seemed to correlate inversely with that of the necrotic area. UCP2 expression was observed, even in nonischemic lobes with similar intralobular heterogeneity.ConclusionsUCP2 was induced in hepatocytes after warm I/R. Although the primitive role of UCP2 expression may be cytoprotective in nature, its actual protective effect in hepatic I/R may be minimal

HMGB1 in ischemic and non-ischemic liver after selective warm ischemia/reperfusion in rat

High mobility group box 1 (HMGB1) acts as an early mediator in inflammation and organ injury. Ischemia reperfusion (I/R) injury induces HMGB1 translocation and expression in ischemic areas. However, it is unknown whether selective warm liver I/R injury also induces the expression of HMGB1 in non-ischemic lobes. The present study aimed to test the hypothesis that selective liver I/R injury also causes HMGB1 translocation and up-regulates its expression in non-ischemic liver areas. In the present study, selective I/R injury was induced by clamping the median and left lateral liver lobes for 90min followed by 0.5, 6 and 24h reperfusion. We used male inbred Lewis rats; six animals for each point in time and six animals for the normal control group. Selective hepatic I/R injury induced morphological changes not only in ischemic lobes but also in non-ischemic lobes. HMGB1 translocation and expression was increased in a time-dependent manner in the ischemic lobes, and increased in with delayed onset in the non-ischemic lobes. Serum HMGB1 levels were increased after reperfusion. Furthermore, liver I/R injury up-regulated the expression of HMGB1 receptors (Toll-like receptor 4 and receptor for advanced glycation end products and pro-inflammatory cytokines (Tumor necrosis factor-alpha and interleukin-6) in both ischemic lobes, however, the up-regulation of these cytokines was more prominent in the ischemic lobes. In conclusion, selective warm I/R induces a substantial "sympathetic/bystander" effect on the non-ischemic lobes in terms of HMGB1 translocation and local cytokine production.

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.

Effects of isoflurane preconditioning on expression of TLR4 and MyD88 during focal cerebral ischemiareperfusion in rats

Objectiye To investgate the effects of isoflurane preconditioning on expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) during focal cerebral ischemia-reperfusion (IR) in rats. Methods Thirty male SD rats weighing 250-300 g were randomly divided into 3 groups ( n = 10 each):sham operation group (group S);focal cerebral IR group and isoflurane preconditioning group (group IP). The animals were anesthetized with intraperitoneal pentobarbital 40 mg/kg. In group IR and IP a nylon thread with rounded tip was inserted into right internal jugular vein and threaded cranially until resistance was met. Mid-cerebral artery was occluded (MCAO) for 2 h followed by 24 h reperfusion. In group IP the animals inhaled 2% isoflurane98 % O2 for 1 h once a day for 5 consecutive days at 24 h before MCAO. Neurologic function was assessed and scored and cerebral infarct volume was measured at 24 h of reperfusion. The animals were sacrificed at 24, 48 and 72 h of reperfusion respectively. The right ischemic frontal lobes were removed for determination of TLR4, MyD88and NF-κB expression by Western blot analysis. Results MCAO significantly worsened neurologic function. The neurologic function deficit scores were significantly increased and the TLR4, MyD88 and NF-κB expression were significantly up-regulated in group IR as compared with group S (P ＜ 0.05). Isoflurane preconditioning significantly decreased cerebral infarct volumes and neurologic function deficit scores and down-regulated the expression of TLR4, MyD88 and NF-κB in group IP as compared with group IR ( P ＜ 0.05). Conclusion Isoflurane preconditioning can reduce inflammatory response and focal cerebral IR injury by down-regulating the expression of TLR4and Myd88. Key words: Isoflurane; Ischemic preconditioning; Toll-like receptor 4; Myeloid differentiation factor 88; Reperfusion injury; Brain

Oxidative Stress in the Ischemic and Non-Ischemic Parts of the Rat Liver after Two-Thirds Ischemia/Reperfusion

Rat liver was subjected to two-thirds warm ischemia for 45 min and reperfusion (I/R) to evaluate the resulting oxidative stress. The plasma alanine aminotransferase and aspartate aminotransferase activities were significantly higher than those in the sham group 1.5-24 h after I/R, showing extensive liver cell death. The level of oxidative stress was compared between the ischemic and non-ischemic regions based on the change in antioxidative vitamins C and E. The vitamin C level was significantly decreased during I/R in both the ischemic and non-ischemic regions 0, 1.5, 3, 6, 12, and 24 h after the start of reperfusion, showing enhanced oxidative stress even in the non-ischemic lobules. This decrease of vitamin C in the ischemic region was significantly higher than that in the non-ischemic lobules, while the vitamin E content was decreased only in the ischemic lobes, demonstrating higher oxidative stress in the ischemic region than that in the non-ischemic region. Early transient activation of cytoprotective extracellular signal-related kinase (ERK) was apparent in both the ischemic and non-ischemic lobules, reflecting oxidative stress in both regions. Early transient activation of c-Jun NH(2)-terminal kinase (JNK) was only apparent in the ischemic region, corresponding to extensive oxidative stress and liver cell death. These results demonstrate that significant oxidative stress was induced, but that JNK leading to cell death was not activated in the non-ischemic part of the liver.

Proteomic Analysis of Hepatic Ischemia and Reperfusion Injury in Mice

Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery. I/R injury induces serious hepatic dysfunction and failure. In this study, we identified proteins that were differentially expressed between sham and I/R injured livers. Animals were subjected to hepatic ischemia for 1 hr and were sacrificed at 3hr after reperfusion. Serum ALT and AST levels were significantly increased in I/R-operated animals compared to those of sham-operated animals. Ischemic hepatic lobes of I/R-operated animals showed the hepatic lesion with unclear condensation and sinusoidal congestion. Proteins from hepatic tissue were separated using two dimensional gel electrophosresis. Protein spots with a greater than 2.5-fold change in intensity were identified by mass spectrometry. Among these proteins, glutaredoxin-3, peroxiredoxin-3, glyoxalase I, spermidine synthase, dynamin-1-like protein, annexin A4, eukaryotic initiation factor 3, eukaryotic initiation factor 4A-I, 26S proteasome, proteasome alpha 1, and proteasome beta 4 levels were significantly decreased in I/R-operated animals compared to those of sham-operated animals. These proteins are related to protein synthesis, cellular growth and stabilization, anti-oxidant action. Moreover, Western blot analysis confirmed that dynamin-1-like protein levels were decreased in I/R-operated animals. Our results suggest that hepatic I/R induces the hepatic cells damage by regulation of several proteins.

Formation of Collateral Circulation Within Hepatic Parenchyma After Recurrent Hepatic Artery Thrombosis and Thrombolysis Early After Liver Transplantation: Case Report

The patient was a 36-year-old man with liver cirrhosis secondary to chronic hepatitis B. Because the donor’s right hepatic artery (RHA) originated from superior mesenteric artery, a continuous end-to-end anastomosis of RHA with splenic artery was performed (6-0 prolene). Then, the confluence of proper hepatic artery and gastroduodenal artery of the recipient was anastomosed with the graft’s celiac trunk in a continuous end-to-end manner (6-0 prolene). Hepatic artery thrombosis (HAT) at anastomotic stoma was confirmed by angiography on postoperative day (POD) 5. After revascularization by injection of urokinase (150,000 U) directly into the thrombus, continuous administration of urokinase (100,000 U, q12h), argatroban (2.0 mg/hr), and papaverine (30 mg, every 12 hours [q12h]) was performed at anastomotic stoma. Twenty hours later, hemorrhage of anastomotic stoma occurred, which gradually ceased after stopping the thrombolysis and anticoagulation therapy. Successful thrombolysis with urokinase (100,000 U) was performed on POD 7 and 8, and continuous anticoagulation of argatroban (2.0 mg/hr) and papaverine (30 mg, q12h) was given without urokinase in case of hemorrhage. RHA was occluded on POD 9. Its revascularization was performed by urokinase (100,000 U). Ten hours later, left hepatic artery (LHA) was occluded. After the same management, argatroban (1.5 mg/hr) and papaverine (30 mg, q12h) were given combined with urokinase (100,000 U, q12h). The occlusion of LHA with bleeding at anastomotic stoma occurred on POD 11. After revascularization with urokinase (100,000 U), argatroban (1.0 mg/hr) and papaverine (30 mg, q12h) were continuously infused, and the hemorrhage was controlled soon. Although LHA was occluded again on POD 12, collateral circulation derived from RHA supplied the ischemic left hepatic lobe. Thrombolysis was not performed this time. Even though LHA was not recanalized from then on, the visualization of RHA and the collateral arteries was fine all along. The catheter remained in the hepatic artery for 27 days. The collateral vessels, including some thick trunks, were abundant in left hepatic parenchyma 1 month posttransplant (Fig. 1). Liver function was normal during thrombolysis therapy and no necrotic foci were found in hepatic parenchyma by computed tomography scanning. The patient was discharged on POD 50 and was in excellent health during the 12-month follow-up.FIGURE 1.: Angiograms at different time points after liver transplantation. (1) Complete occlusion of hepatic artery by thrombus at anastomotic stoma (postoperative day [POD] 5). (2) Relatively satisfactory revascularization with totally 150,000 U of urokinase (POD 5). (3) Bleeding at anastomotic stoma (POD 6). (4) Occlusion of right hepatic artery (RHA) with patent left hepatic artery (LHA) (POD 9). (5) Occlusion of LHA with patent RHA (POD 9). (6) Occlusion of LHA combined with bleeding at anastomotic stoma (POD 11). (7) Collateral circulation from RHA to left hepatic lobe (POD 12). (8) Plentiful collateral arteries in left hepatic parenchyma, including some thick trunks (POD 32).It was reported that HAT was identified again in three orthotopic liver transplantation patients after hepatic artery revascularization and immediate second thrombolysis with urokinase was performed successfully (1). We herein first described a patient who underwent HAT as many as seven times. Successful revascularization with urokinase was achieved each time except for the last one when urokinase was not used for the formation of collateral circulation. Argatroban, a synthetic direct thrombin inhibitor (2), was introduced for the treatment of HAT in this case. When argatroban was used for anticoagulation without urokinase, there was no bleeding, which showed its safety. It needs to be further investigated whether argatroban can be routinely used to prevent HAT after liver transplantation. Different from the cases previously reported (3, 4), the collateral circulation in this case ran within the hepatic parenchyma from RHA to left hepatic lobe. It appeared as early as 2 weeks after transplantation, hence saved the ischemic region. We speculated that the fact that the time required to form collateral circulation in our case was shorter than that in other cases was likely to be attributed to the highly developed vascular network in liver. Bing Han Chang Liu Department of Hepatobiliary Surgery The First Affiliated Hospital of Medical College Xi’an Jiaotong University Shaanxi Province, China Hongyan Tian Yamin Liu Department of Cardiopathy and Peripheral Angiopathy The First Affiliated Hospital of Medical College Xi’an Jiaotong University Shaanxi Province, China Liang Yu Xuemin Liu Bo Meng Department of Hepatobiliary Surgery The First Affiliated Hospital of Medical College Xi’an Jiaotong University Shaanxi Province, China Hongmei Wan Department of Cardiopathy and Peripheral Angiopathy The First Affiliated Hospital of Medical College Xi’an Jiaotong University Shaanxi Province, China Naiying Shen Haitao Zhu Yi Lu Department of Hepatobiliary Surgery The First Affiliated Hospital of Medical College Xi’an Jiaotong University Shaanxi Province, China

Human Heat Shock Protein 27 Overexpressing Mice Are Protected Against Hepatic Ischemia and Reperfusion Injury

Hepatic ischemia reperfusion injury (IRI) is a major clinical problem during the perioperative period and occurs frequently after major hepatic resection or liver transplantation. Our laboratory previously demonstrated that exogenous A1 adenosine receptor activation protects against renal IRI by upregulation and phosphorylation of heat shock protein 27 (HSP27). This study used mice overexpressing human HSP27 (huHSP27 OE) to determine whether these mice are protected against liver IRI. After hepatic IR, the huHSP27 OE mice had significant protection against liver injury (reduced alanine transferase) and necrosis (hematoxylin-eosin staining) compared with the HSP27 WT mice. The huHSP27 OE mice also showed less induction of proinflammatory messenger RNA MIP-2, reduced neutrophil infiltration, and decreased apoptosis (caspase 3 fragmentation and DNA laddering) compared with the HSP27 WT mice. Finally, the huHSP27 OE mice showed significantly less disruption of filamentous actin in hepatocytes and bile canaliculi of the ischemic lobes compared with the HSP27 WT mice. Depletion of Kupffer cells with gadolinium chloride provided significant protection against liver IRI in HSP27 WT mice but not in huHSP27 OE mice suggesting that the overexpression of huHSP27 in the Kupffer cells may be responsible for the hepatic protection observed in huHSP27 OE mice. Our results show that the overexpression of huHSP27 in Kupffer cells of the liver may be responsible for the protection against hepatic IRI in vivo by reducing necrosis and apoptosis and by stabilizing F-actin with subsequent reductions in inflammation and proinflammatory neutrophil infiltration. Harnessing the mechanisms of cytoprotection with HSP27 may lead to new therapies for the management of perioperative hepatic IRI.

Short Passive Cooling Protects Rats During Hepatectomy by Inducing Heat Shock Proteins and Limiting the Induction of Pro-Inflammatory Cytokines

Prolonged hepatic warm ischemia during surgery remains a significant problem, particularly in the setting of liver resection and reduced remaining liver mass. The goal of the present study is to evaluate the effect of passive cooling caused by exposure to ambient conditions on hepatic injury in rats during warm ischemia followed by hepatectomy. The left and median lobes of male rats were exposed to 75 min of ischemia under either normothermic (37 degrees C) or mildly hypothermic (34 degrees C) conditions. After 75 min of ischemia, the right lobe was resected, leaving the animal with only the remaining ischemic lobes. Animals were allowed to survive indefinitely or sacrificed at 4 h after reperfusion for determination of injury and inflammatory gene expression. Survival was already markedly higher in mildly hypothermic rats than normothermic rats at 24 h. Short passive cooling for the time course of the ischemic event significantly increased the hepatic induction of heat shock proteins 70 and 32 (both 3-fold versus normothermia, P<0.05) in response to ischemia/reperfusion whereas it significantly decreased the induction of tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-2 (MIP-2) in the liver. Biochemical markers of hepatic injury were significantly lower in the passive cooling group than in normothermic animals: aspartate aminotransferase (AST) serum concentrations were 9277+/-3461IU/L versus 15106+/-4104IU/L (P<0.01), and alanine aminotransferase (ALT) levels 5986+/-2246IU/L versus 9429+/-3643IU/L (P<0.01). We demonstrated in a clinically relevant model of hepatic ischemia/reperfusion that mild hypothermia significantly reduces hepatic injury and improves survival.

Mouse model of liver ischemia and reperfusion injury: method for studying reactive oxygen and nitrogen metabolites in vivo

The mouse model of liver ischemia and reperfusion injury has proven to be valuable for our understanding of the role that reactive oxygen and nitrogen metabolites play in postischemic tissue injury. This methods paper provides a detailed protocol for inducing partial liver ischemia followed by reperfusion. Liver ischemia is induced in anesthetized mice by cross-clamping the hepatic artery and portal vein for varying lengths of time, resulting in deprivation of blood flow to approximately 70% of the liver. Restoration of blood flow to the ischemic lobes enhances superoxide production concomitant with a rapid and marked decrease in the bioavailability of nitric oxide, resulting in alterations in the redox state of the liver in favor of a more oxidative environment. This hepatocellular oxidative stress induces the activation of oxidant-sensitive transcription factors followed by the upregulation of proinflammatory cytokines and mediators that ultimately lead to liver injury. This model can be induced in any strain or sex of mouse and requires 1–2 months of practice to become proficient in the surgery and animal manipulation. The roles of various reactive metabolites of oxygen and nitrogen may be evaluated using genetically engineered mice as well as selective molecular, cellular, and/or pharmacological agents.

Significance of rosiglitazone inhibiting TLR4 expression in partial hepatic ischemia/reperfusion of mice

The effect of rosiglitazone as the ligand of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibiting the TLR4 expression in ischemic lobes in partial hepatic ischemia/reperfusion injury (IRI) in BALB/C mice and the action of rosiglitazone inhibiting the TLR4 receptor-mediated inherent immune response were investigated. The model of the mouse partial hepatic ischemia/reperfusion injury was established. All the animals were randomly divided into 3 groups: rosiglitazone group, vehicle (dimethylsulphoxide, DMSO) group and sham operation group. The hepatic samples were collected when mice were sacrificed 0, 2, 4 and 6 h after reperfusion following 1 h ischemia to analyze the acute phase of hepatic IRI. The dynamic expression of TIR4 mRNA was detected quantitatively by real-time-PCR, and the levels of TNF-alpha, IL-10 and ALT in portal vein were determined in all groups. After restoration of blood supply, the expression of TLR4 mRNA in ischemic lobes was detected in 0, 2, 4 and 6 h after reperfusion following 1 h ischemia in rosiglitazone group and vehicle group. The most intensive expression of TLR4 mRNA was present at 4 h after reperfusion in ischemic lobes in vehicle group. As compared with vehicle group, the expression of TLR4 mRNA in ischemic lobes in rosiglitazone group was significantly decreased at 4 h after reperfusion. The level of IL-10 in portal vein was markedly up-regulated in rosiglitazone group as compared with vehicle group. Contrarily, the levels of TNF-alpha and ALT in portal vein were markedly down-regulated in rosiglitazone group as compared with vehicle group at every time point in mouse partial hepatic IRI model. Rosiglitazone could alleviate the hepatic IRI by inhibiting TLR4 receptor-mediated inherent immune response.

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.