Ischemic Liver Lobes Research Articles

ICAM-1 Signal Transduction in Cells Stimulated with Neutrophil Elastase

Neutrophil elastase, which enhances intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells, plays an important role in ischemia/reperfusion injury. Here, we investigated signal transduction of ICAM-1 expression in endothelial cells stimulated by neutrophil elastase. Pretreatment of animals with the neutrophil elastase inhibitor, ONO-5046.Na significantly decreased the number of neutrophils or Mac-1(+) (CD11b/CD18) cells in ischemic liver lobes after reperfusion. ICAM-1 expression in the rat endothelial cell line (WK-5) was significantly upregulated after stimulation with neutrophil elastase, but this reaction was inhibited by the neutrophil elastase inhibitor ONO-5046.Na. ICAM-1 mRNA expression, which is induced by neutrophil elastase in a dose-dependent manner, was repressed by the alpha1-protease inhibitor. ICAM-1 expression, stimulated by neutrophil elastase, was partially reduced by a diacylglycerol kinase inhibitor and protein kinase C inhibitor, but was completely inhibited by a phospholipase C inhibitor, cytosolic Ca(2+) chelator, calmodulin antagonist, and nuclear transcription factor kappa B inhibitor. Binding of (125)I-neutrophil elastase to WK-5 cells was competitively inhibited by the addition of unlabeled neutrophil elastase. The neutrophil elastase inhibitor significantly reduces ICAM-1 expression and Mac-1(+) cell accumulation in ischemic liver lobes after reperfusion. Neutrophil elastase stimulates ICAM-1 expression in endothelial cells by intracellular signal transduction via activation of diacylglycerol kinase, protein kinase C, phospholipase C, Ca(2+)-calmodulin, and nuclear transcription factor kappa B.

Vascular Endothelial Growth Factor Antagonist Modulates Leukocyte Trafficking and Protects Mouse Livers against Ischemia/Reperfusion Injury

Although hypoxia stimulates the expression of vascular endothelial growth factor (VEGF), little is known of the role or mechanism by which VEGF functions after ischemia and reperfusion (I/R) injury. In this report, we first evaluated the expression of VEGF in a mouse model of liver warm ischemia. We found that the expression of VEGF increased after ischemia but peaked between 2 and 6 hours after reperfusion. Mice were treated with a neutralizing anti-mouse VEGF antiserum (anti-VEGF) or control serum daily from day -1 (1 day before the initiation of ischemia). Treatment with anti-VEGF significantly reduced serum glutaminic pyruvic transaminase levels and reduced histological evidence of hepatocellular damage compared with controls. Anti-VEGF also markedly decreased T-cell, macrophage, and neutrophil accumulation within livers and reduced the frequency of intrahepatic apoptotic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells. Moreover, there was a reduction in the expression of pro-inflammatory cytokines (tumor necrosis factor-alpha and interferon-gamma), chemokines (interferon-inducible protein-10 and monocyte chemoattractant protein-1) and adhesion molecules (E-selectin) in parallel with enhanced expression of anti-apoptotic genes (Bcl-2/Bcl-xl and heme oxygenase-1) in anti-VEGF-treated animals. In conclusion, hypoxia-inducible VEGF expression by hepatocytes modulates leukocyte trafficking and leukocyte-induced injury in a mouse liver model of warm I/R injury, demonstrating the importance of endogenous VEGF production in the pathophysiology of hepatic I/R injury.

Fas Ligand Expression Following Normothermic Liver Ischemia-Reperfusion

The aim of this study was to evaluate the role of the pro-apoptotic molecule Fas Ligand (FasL) in 120 min normothermic ischemia-reperfusion (I-R) induced apoptosis in rat liver treated or not with Z-Asp-cmk caspase inhibitor. Rats were divided into two groups: group 1, control, PBS administration; group 2, Z-Asp-cmk treatment. Z-Asp-cmk was injected intravenously, 2 min before induction of 120 min of normothermic liver ischemia. Immunohistochemical detection of apoptotic liver cells was carried out using the TUNEL method. Fas and FasL expression were measured by qualitative reverse transcription polymerase chain reaction (RT-PCR), Northern and western blot, and by immunofluorescence labeling, in ischemic and non-ischemic liver lobes at different times after reperfusion. FasL mRNA and protein expression were increased in ischemic liver, while Fas receptor mRNA levels remained unchanged. Pre-treatment of rats with Z-Asp-cmk caspase inhibitor reduced liver apoptosis, but did not modify FasL mRNA levels. These results suggest that the pro-apoptotic molecule FasL is involved in the induction of liver apoptosis following I-R.

Cytoprotection by bcl-2 gene transfer against ischemic liver injuries together with repressed lipid peroxidation and increased ascorbic acid in livers and serum.

The maximum gene exhibition was shown to be achieved at 48 h after transfection with human bcl-2 (hbcl-2) genes built in an SV40 early promoter-based plasmid vector and HVJ-liposome for cultured rat hepatocytes. The similar procedure of hbcl-2 transfection was therefore conducted for livers in rats via the portal vein, and after 48 h followed by post-ischemic reperfusion (I/R) operation for some hepatic lobes. The I/R-induced hepatic injuries were in situ observed as both cell morphological degeneration and cellular DNA strand cleavages around capillary vessels of the ischemic liver lobes as detected by HE stain and TUNEL assay, and were biochemically observed as release of two hepatic marker enzymes AST and ALT into serum. All the I/R-induced injuries examined were appreciably repressed for rats transfected with hbcl-2; hbcl-2 was expressed in hepatocytes around the capillaries of ischemic regions such as the median lobe and the left lobe, but scarcely around those of non-ischemic regions. Thus cytoprotection against I/R-induced injuries may be attributed to the I/R-promoted expression of transferred hbcl-2 genes. The possibility was examined firstly by methylphenylindole method, which showed that I/R-enhanced lipid peroxidation in the reference vector-transfected livers were markedly repressed in the hbcl-2-transfected livers. Contents of ascorbic acid (Asc) in serum and livers of hbcl-2-transfected rats were enriched, unexpectedly, versus those of non-transfected rats, and were as abundant as 1.90-fold and 1.95- to 2.60-fold versus those in the pre-ischemic state, respectively. After I/R, an immediate decline in serum Asc occurred in hbcl-2-transfectants, and was followed by prompt restoration up to the pre-ischemic Asc levels in contrast to the unaltered lower Asc levels in non-transfectants except a transient delayed increase. Hepatic Asc contents were also diminished appreciably at the initial stage after I/R in the ischemic lobes of hbcl-2-transfectants, which however retained more abundant Asc versus non-transfectants especially at the initial I/R stage when scavenging of the oxidative stress should be most necessary for cytoprotection. The results showed a close correlation between cytoprotection by exogenously transferred hbcl-2 and repressive effects on the lipid peroxidation associated with Asc consumption or redistribution.

ANTI-APOPTOTIC PI3-K/PKB PATHWAY IS INACTIVATED AFTER NORMOTHERMIC LIVER ISCHEMIA-REPERFUSION

A20 Aims: The aim of this study was to evaluate the role of the protein kinase B (PKB), phosphatidyl inositol-3 kinase (PI3-K) and BAD in normothermic ischemia-reperfusion (I-R) induced apoptosis in rat liver treated or not with Z-Asp-cmk caspase inhibitor. Methods: Rats were divided into two groups: group 1, control, PBS administration; group 2, Z-Asp-cmk treatment. Z-Asp-cmk was injected intravenously, 2 min prior to induction of 120 min of normothermic liver ischemia. PI3-K, PKB and BAD activities were measured in ischemic and non-ischemic lobes at different times after reperfusion. Immunohistochemical detection of apoptotic liver cells was carried out using the TUNEL method. Results: In control rats, PI3-K and PKB activities were inhibited in ischemic rat liver lobes. BAD phosphorylation was abolished after liver I-R. PKB activity was not modified by pre-treatment with Z-Asp-cmk caspase inhibitor whereas liver apoptosis was reduced. Conclusions: These results suggest that induction of apoptosis during I-R liver injury is triggered by the inactivation of the anti-apoptotic PI3-K/PKB pathway.

Intermittent hepatic ischemia-reperfusion minimizes liver metastasis in rats

Background. Surgical stresses, including hepatic ischemia-reperfusion (I/R), promote cancer growth and metastasis. We have reported that continuous hepatic I/R increases liver damage and promoted liver metastasis from colon cancer, whereas intermittent I/R causes less liver damage. We therefore examined whether intermittent I/R could reduce liver metastasis in a rat model. Materials and method. Adult male Fischer rats was divided between three groups: group A (control), which received laparotomy for 120 min with no liver ischemia; group B (continuous I/R), which received 60 min of 70% partial liver ischemia followed by 60 min of reperfusion; and group C (intermittent I/R), which received 15 min of 70% ischemia and 15 min of reperfusion, repeated four times. Just before closing the abdomen, all animals were inoculated intrasplenically with rat colon adenocarcinoma cells (RCN-H4). Tumor nodules on the liver surface were counted 3 weeks later. In addition, expression of E-selectin mRNA in liver was examined at 1, 3, and 6 h after completing I/R by a reverse transcription-polymerase chain reaction. Results. Continuous I/R (B) greatly promoted liver metastasis in both ischemic and nonischemic liver lobes, whereas intermittent I/R (C) showed significantly fewer metastasis than group B in both lobes. Significantly less E-selectin mRNA was expressed in group C than in group B. Conclusions. Intermittent I/R limits expression of E-selectin mRNA and liver metastasis. Intermittent hepatic I/R is less stressful than continuous I/R, minimizing liver metastasis by colon cancer cells through avoidance of E-selectin up-regulation.

Ischemia at 4 degrees C: a novel mouse model to investigate the effect of hypothermia on postischemic hepatic microcirculatory injury.

Hypothermia of the ischemic organ at 4 degrees C protects hepatic microcirculation from ischemia-reperfusion (IR) injury. The effect of hypothermia during ischemia was investigated in animal models using liver transplantation and storage of the harvested organ in cold preservation solutions. No investigation of the isolated influence of hypothermia at 4 degrees C of the ischemic organ on hepatic IR injury exists, due to the lack of an appropriate animal model. Therefore, the aim of our present study was to develop such a model using intravital video fluorescence microscopy (IVM). In C57BL/6 mice, a reversible isolated ischemia of the left liver lobe was induced for 90 min, followed by 240 min of reperfusion. The temperature of the ischemic organ was adjusted to either 4 degrees C or 37 degrees C by superfusion with 0.9% NaCl. Sham-operated animals without IR served as controls. The hepatic microcirculation was analyzed using IVM at 30 min and 240 min after reperfusion by quantifying sinusoidal perfusion and leukocyte-endothelial cell interaction in postsinusoidal venules. At the end of the experiment, blood and tissue samples were taken for measurement of liver enzyme activities and light and electron microscopy. Mean arterial pressure and body temperature were kept constant throughout the experiment, while the temperature of the ischemic liver lobe was adjusted to predefined levels. After normothermic ischemia, hepatic microvascular perfusion was significantly impaired compared with sham-operated animals. Perfusion failure was significantly reduced in hypothermic livers and did not differ from livers of the sham-group. Liver enzyme activities in the normotherimic group were significantly higher than in the sham and hypothermic groups. Light and electron microscopy revealed severe histological alterations at 37 degrees C ischemia, whereas at 4 degrees C ischemia only minimal lesions were encountered. Our novel model allows for isolated adjustment of ischemic liver lobe temperature without changing body temperature and systemic macrohemodynamic parameters. Hypothermia at 4 degrees C largely attenuates postischemic microvascular perfusion injury of the liver.

Role of endogenous nitric oxide in TNF-alpha and IL-1beta generation in hepatic ischemia-repefusion.

In the present study, we examined the role of nitric oxide (NO) in early-response cytokine production by using a rat model of hepatic ischemia-reperfusion (HI/R). The left and median lobes of the liver were subjected to 30 min of ischemia, followed by 4 h of reperfusion. Group I and II rats were sham-operated controls that received saline (vehicle) or N(W)-nitro-L-arginine methylester (L-NAME) (10 mg/kg, iv); group III and IV rats were subjected to HI/R and received vehicle or L-NAME (10 mg/kg, iv, 10 min before reperfusion), respectively. Administration of L-NAME to rats subjected to I/R resulted in a fourfold decrease in plasma NO levels, accompanied by a marked increase of plasma alanine aminotransferase (ALT) activity relative to group III. These changes in group IV were associated with elevation of superoxide generation in ischemic liver lobes by 2.1-fold and circulating leukocyte number by 1.42-fold, compared with group III. Normalized for expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger ribonucleic acid (mRNA), expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) mRNA in ischemic liver of group IV was augmented by 207% and 175% compared with Group III. The expression of (iNOS) mRNA was also increased (223%) relative to group III. Moreover, in group IV, plasma TNF-alpha levels at 4 h of reperfusion and IL-1beta levels at 90 min and 4 h of reperfusion were significantly increased compared with group III. No statistically significant changes were observed between groups I and II in plasma ALT activity, plasma NO levels, circulating leukocyte counts, superoxide generation in the ischemic lobes of liver, and plasma TNF-a and IL-1beta concentrations. The observed enhancement of I/R injury by L-NAME is consistent with the hypothesis that endogenous NO down-regulates TNF-alpha and IL1beta generation, thereby decreasing HI/R injury.

A caspase inhibitor fully protects rats against lethal normothermic liver ischemia by inhibition of liver apoptosis.

Apoptosisis activated during the early phase of reperfusion after liver ischemia and after liver transplantation in animals. However, the molecular basis of ischemia-induced cell death remains poorly understood. In this study we show that hepatocytes from ischemic liver lobes undergo apoptosis after reperfusion. In vivo pretreatment of rats with a specific inhibitor of caspases abrogates the apoptotic response in ischemic liver lobes. Inhibition of apoptosis can be accounted for by total inhibition of caspase activation as assessed in an enzymatic assay and by specific affinity labeling. Treatment with a caspase inhibitor fully protects rats from death induced by ischemia/reperfusion. These findings indicate that liver injury after ischemia/reperfusion can be prevented by inhibition of caspases. Thus, caspase inhibitors may have important therapeutic implications in liver ischemic diseases and after liver transplantation.

Role of P-selectin expression in hepatic ischemia and reperfusion injury.

Researchers have shown that reperfusion of ischemic tissues initiates a complex series of reactions that paradoxically injure tissues. Although several mechanisms have been proposed to explain the pathobiology of ischemic/reperfusion (I/R) injury, much attention has focused on adhesion molecules. Our research is intended to show the kinetics of P-selectin in the liver in response to I/R injury. Left-lobar hepatic ischemia was induced for 30 min in 35 C57BL-6 mice and 20 P-selectin-deficient (K-O) mice. P-selectin expression was measured in these mice at 20 min, 2, 5, 12 and 24 h reperfusion times, as well as in control and sham animals. The animals were injected with radio-labeled P-selectin monoclonal antibody and the organs were harvested for counts/g tissue, expressed as the percentage injected dose. Serum liver enzymes were measured and pathological sections of ischemic and control livers were performed. The unpaired t-test was used for statistical analysis. P-selectin expression showed two peaks in this animal model. The first peak was at 20 min and the second peak at 5 h of reperfusion (p < 0.001). We documented an 8-fold increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels 10 h following I/R injury. Pathological specimens showed periportal necrosis consistent with an ischemic event. P-selectin K-O mice showed no up-regulation as a separate control group, and the liver enzymes were significantly lower than the wild-type mice at 10 h (p < 0.001). P-selectin has a bimodal expression following hepatic I/R injury. The first peak is attributed to the Weibel-Palade bodies and the second to new translational P-selectin. We noted no difference in the up-regulation of P-selectin in the ischemic and non-ischemic liver lobes in the same animal.

Intercellular adhesion molecule 1 (ICAM-1) expression and its role in neutrophil-induced ischemia-reperfusion injury in rat liver

The potential role of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of reperfusion injury was investigated in male Fischer rats subjected to 45 min of hepatic ischemia and 24 h of reperfusion. ICAM-1 mRNA levels increased during ischemia in the ischemic liver lobes; however, during reperfusion mRNA levels increased in both the ischemic and nonischemic lobes. Immunohistochemical evaluation indicated ICAM-1 expression only on sinusoidal lining cells in controls; ischemia-reperfusion enhanced ICAM-1 expression in the sinusoids and induced some expression on hepatocytes. The monoclonal anti-ICAM-1 antibody 1A29, but not an immunoglobulin G control antibody, administered at 1 h and 8 h of reperfusion (2 mg/kg) significantly attenuated liver injury as indicated by 51% lower plasma alanine aminotransferase activities and 32-36% less hepatic necrosis at 24 h without affecting reactive oxygen formation by Kupffer cells and hepatic neutrophils. Although 1A29 reduced neutrophil extravasation in a glycogen peritonitis by 60%, the antibody had no significant effect on hepatic neutrophil infiltration during reperfusion. These data suggest that ICAM-1 plays a significant role during the neutrophil-dependent injury phase after hepatic ischemia and reperfusion and therefore blocking this adhesion molecule may have therapeutic potential against postischemic acute liver failure.

Patterns of RNA and Protein Synthesis in Post-Ischemic Livers

The re-establishment of the blood supply to a formerly ischemic liver lobe, before the "point of no return" of the tissue is reached, induces a series of changes in protein and RNA metabolism that are functional to the repair of the damage suffered by the cells. Among these events there is the increase in synthesis of a group of proteins known as heat-shock (or stress) proteins, which are also induced in liver cells by different kinds of oxidative stress. The increase in synthesis of these proteins is largely due to the activation of their genes: some of these genes are also activated in cells stimulated to grow. These observations suggest a link between oxidative stress, repair of cell damage and cell multiplication.

Changes in cytoplasmic and mitochondrial enzymes in rat liver after ischemia followed by reperfusion

The behavior of cytoplasmic and mitochondrial enzymes has been studied in rat liver at 1, 5, and 24 hr after 60 min of ischemia using histochemical methods. This period of ischemia resulted 24 h after ischemia in liver cell necrosis in about 15% of the volume of the ischemic liver lobes. As early as after 1 hr reperfusion lactate dehydrogenase (LDH, cytoplasm) activity decreased in a certain proportion of the liver parenchymal cells, whereas glutamate dehydrogenase (GDH, mitochondrial matrix) activity started to decrease after 5 hr reperfusion; the activities of mitochondrial membrane enzymes, monoamine oxidase and succinate dehydrogenase, did not decrease before 24 hr of reperfusion. It has been concluded that the early decrease in LDH activity is caused by leakage into the blood and reflects reversible damage; when this decrease is accompanied by a decrease in GDH activity irreversible liver cell damage is assumed. Diminished activity of mitochondrial membrane enzymes, due to leakage and denaturation, is observed when real necrosis can be assessed.