Regulation of pro-inflammatory and anti-inflammatory cytokine responses by Kupffer cells in endotoxin-enhanced reperfusion injury after total hepatic ischemia

The effects of Kupffer cells on cytokine responses in endotoxin-enhanced reperfusion injury after total hepatic ischemia were investigated in this study. Male rats pretreated with either normal saline solution (NS group) or gadolinium chloride (GdCl(3)) to inhibit Kupffer cell function (GC group) were subjected to 60 min of hepatic ischemia. These animals received either normal saline solution or sublethal doses of endotoxin (1 mg/kg) at reperfusion. In the NS group, endotoxin administration induced an enhanced tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 production 1 h after reperfusion with a subsequent peak of macrophage inflammatory protein-2 (MIP-2) levels, which resulted in a 7-day survival rate of 30%. Despite endotoxin administration, GdCl(3) pretreatment significantly suppressed TNF-alpha and increased interleukin-10 production 1 h after reperfusion, which led to a decline in MIP-2 production and amelioration of functional and structural liver damage with a 7-day survival rate of 80%. Augmented pro-inflammatory and anti-inflammatory cytokine responses by Kupffer cells were associated with endotoxin-enhanced reperfusion injury after hepatic ischemia. Kupffer cell blockade has a potential to attenuate the insult via modulation of cytokine responses.     

Role of cytokines in hepatic ischemia and reperfusion injury]

The liver is an organ with abundant blood flow, consisting of hepatic arterial and portal blood flow. The viability of liver tissue depends on the condition of the hepatic microcirculation which is controlled by hepatic sinusoidal lining cells. Hepatic ischemia and reperfusion (HIR) injury is inevitable in surgical procedures for liver trauma and hepatectomy as well as liver transplantation. Reperfusion through an ischemically damaged organ enhances the tissue injury. Cytokines are pivotal factors in neutrophil-mediated liver injury following HIR, while various other mediators are involved in this insult. Advances in molecular biology have allowed the identification of various cytokines. Inflammatory cytokines such as TNF-alpha are associated with the induction of cellular adhesion molecules and hepatic microcirculatory impairment based on neutrophil-vascular endothelial cell interaction. Members of the chemokine family such as IL-8, CINC, MIP-2, and MCP-1 are involved in neutrophil infiltration in the liver and remote organs. Since each cytokine has a wide variety of actions and interacts' among others' via the cytokine network, their actions in HIR injury have not been determined completely. Kupffer cells have been focused on as a source of cytokine production in HIR injury. Further studies on the mechanisms of cytokine production after HIR and analysis of regulation in the cytokine network would clarify the pathophysiology of HIR injury and the most suitable therapeutic strategy for this insult.     

趋化因子MIP 2在大鼠肝缺血/再灌注损伤后的表达

目的　研究趋化因子巨噬细胞炎性蛋白 -2 (MIP 2 )在肝缺血 /再灌注损伤中 (I/R )的作用。方法　 3 2只大鼠随机分为 4组 ,每组 8只。即假手术 (对照 )组 ,部分肝脏缺血 90min再灌注 3 ,9,2 4h组。用RT PCR法检测肝组织中MIP 2的mRNA表达 ,ELISA法测定血浆中MIP 2蛋白表达 ,萘酚AS D氯醋酸盐酯酶特染技术检测肝组织内中性粒细胞浸润 ,并测定丙氨酸转氨酶 (ALT )。结果　缺血再灌注肝组织中MIP 2mRNA表达高于非缺血肝组织 (P <0 .0 1) ,9h组高于 3h组 (P <0 .0 1) ,2 4h组高于 9h组 (P <0 .0 1) ;MIP 2血浆蛋白表达、肝内中性粒细胞浸润、ALT也与MIP 2mR NA呈一致性的变化。MIP 2mRNA表达、血浆MIP 2蛋白水平与肝组织中性粒细胞浸润呈正相关 ,(r= 0 .88和 0 .83 ,P <0 .0 1)。结论　MIP 2在大鼠肝缺血 /再灌注损伤中作为中性粒细胞的趋化因子之一起着重要的上调作用。     

Hepatic ischemia/reperfusion injury in P-selectin and intercellular adhesion molecule-1 double-mutant mice

Neutrophil adhesion and recruitment represents one of the early cellular events that occur during hepatic ischemia/reperfusion (IR) injury and plays a critical role in determining the extent of tissue damage. The adhesion molecules, such as selectins and intercellular adhesion molecules (ICAM), are important in mediating neutrophil-endothelial cell interactions and neutrophil emigration. The goal of this study was to evaluate the role of P-selectin and ICAM-1 in hepatic IR injury. Male wild-type and P-selectin/ICAM-1-deficient (P/I null) mice underwent 90 minutes of partial hepatic ischemia followed by reperfusion at various time points (0, 1.5, 3, and 6 hours). Reperfusion caused a time-dependent hepatocellular injury in both wild-type and P/I null mice as judged by plasma alanine aminotransferase (ALT) levels and liver histopathology examination. Although ALT levels were slightly lower in the P/I null mice compared with the wild-type mice the differences were not statistically significant. Neutrophil infiltration to the ischemic liver was observed in both mouse groups after 6 hours of reperfusion; however, the infiltration to the midzonal region of the ischemic liver was more pronounced in the wild-type group. This study suggests that hepatocellular injury induced after hepatic IR was independent of P-selectin and ICAM-1 in this model of acute inflammatory tissue injury.     

P-selectin and chemokine response after liver ischemia and reperfusion

BACKGROUND: P-selectin plays a major role in the earliest phase of polymorphonuclear neutrophil recruitment in the hepatic microvasculature after liver ischemia and reperfusion. Leukocyte cytokine chemoattractants (chemokines) cause polymorphonuclear neutrophil activation in ischemia and reperfusion injury. In this study, we examined the role of P-selectin in the production of chemokines in the liver and lung inflammatory response after 90 minutes of warm ischemia. STUDY DESIGN: Thirty-six C57BL/6 mice were subjected to partial liver ischemia for 90 minutes. Three groups of animals were included (n = 12 per group): the sham group, the ischemic control group, and the P-selectin-deficient gene targeted mice group. After 3 hours, we evaluated liver injury measurements, serum chemokines (MIP[macrophage inflammatory protein]-1alpha and MIP-2), liver and lung tissue myeloperoxidase, and liver and lung histology. Statistical analysis included ANOVA, Student-Newman-Keuls', and Kruskal-Wallis Multiple Comparison Z-value tests. RESULTS: P-selectin-deficient mice showed significant decreases in liver enzyme levels (p < 0.05) and marked decreases in serum MIP-1alpha and MIP-2 chemokine determinations (p < 0.05) when compared with ischemic controls. Neutrophil infiltration was significantly ameliorated in the liver (p < 0.05) and markedly decreased in the lung, as reflected by decreased MPO levels. Improved histopathologic features in the liver and lung were observed in the P-selectin-deficient mice group compared with ischemic controls. CONCLUSIONS: Our study confirms the key role of P-selectin in the pathogenesis of liver ischemia and reperfusion and the production of chemokines. P-selectin-deficient animals had improved liver function, decreased neutrophil infiltration, and decreased MIP- 1alpha and MIP-2 responses.     

L-Selectin and chemokine response after liver ischemia and reperfusion

BACKGROUND: L-selectin plays an important role in the early phase of PMNs recruitment in the hepatic microvasculature following liver ischemia and reperfusion (I/R). Leukocyte cytokine chemoattractants (chemokines) cause polymorphonuclear neutrophil (PMN) activation in I/R injury. In this study, we examined the role of L-selectin in the production of chemokines in the liver and lung inflammatory response following 90 min of warm ischemia. STUDY DESIGN: Thirty-six C57BL/6 mice were subjected to partial liver ischemia for a period of 90 min. Three groups of animals were included (n = 12 per group)-sham group, ischemic control, and the ischemic group receiving monoclonal antibody against L-selectin. We evaluated at 3 h: liver injury measurements, serum chemokines (MIP-2 and MIP-1alpha), liver and lung tissue myeloperoxidase (MPO), and liver and lung histology. Statistical analysis included ANOVA, Student-Newman-Keuls', and Kruskal-Wallis multiple comparison Z-value tests. RESULTS: The ischemic group treated with anti-L-selectin showed significant decreases in liver enzyme levels and a marked decrease in serum MIP-2 (P < 0.05) when compared to ischemic controls. No reduction in serum MIP-1alpha was noted; however, neutrophil infiltration was significantly ameliorated in the liver and in the lung, as reflected by decreased MPO levels (P < 0.05). Improved histopathological features were observed in the anti-L-selectin-treated group compared to ischemic controls in the liver and the lung. CONCLUSIONS: Our study suggests an important role for L-selectin in the pathogenesis of liver I/R and the production of chemokines. Anti-L-selectin treatment resulted in improved liver function, decreased neutrophil infiltration, and decreased MIP-2 chemokine response.     

Nonspecific phosphodiesterase inhibition attenuates liver injury in acute endotoxemia

BACKGROUND: Endotoxemia is accompanied by pro-inflammatory cytokine production, generation of reactive oxygen species, and end-organ injury. Pentoxifylline (PTX), a methylxanthine derivative and phosphodiesterase inhibitor, is known for its anti-inflammatory properties, including down-regulation of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha synthesis. Its effects on liver function and hepatic histology following acute endotoxemia have not been investigated fully. We hypothesized that PTX would preserve liver architecture and function after intravenous lipopolysaccharide (LPS) injection. METHODS: Anesthetized Sprague-Dawley rats received an i.v. bolus injection of LPS (5 mg/kg), LPS + PTX (25 mg/kg), or saline (sham). Plasma concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), TNF-alpha, IL-6, and nitrite were measured at different time points after LPS injection. Liver injury was graded according to a scoring system in a blinded fashion from 0 (normal) to 4 (severe) for hepatocellular necrosis, hemorrhage, and parenchymal and sinusoidal inflammatory infiltrates. Neutrophil infiltration was measured by counting myeloperoxidase (MPO)-positive stained cells. Nuclear factor (NF)-kappaB p-65 was measured by counting positive stained nuclei of hepatocytes and Kupffer cells (KC). Inducible nitric oxide synthase (iNOS) was evaluated by positively stained KC. Data are presented as mean +/- SEM. Analysis of variance with p < 0.05 was considered statistically significant. RESULTS: Animals treated with PTX showed a significant reduction in liver injury score and neutrophil infiltration. Treatment with PTX significantly decreased TNF-alpha, IL-6, and the concentrations of AST and ALT when compared to LPS alone. In addition, a significant decrease in NF-kappaB-positive staining in hepatocytes and KC, as well as in KC iNOS immunostaining was observed in PTX-treated animals compared to the LPS group. CONCLUSIONS: Pentoxifylline downregulates the inflammatory response significantly and decreases liver injury in acute endotoxemia.     

Selectin inhibition modulates Akt/MAPK signaling and chemokine expression after liver ischemia-reperfusion.

Tissue damage after ischemia and reperfusion (I/R) is largely caused by the sequelae of neutrophil infiltration. This inflammatory process can be initiated as the result of stroke, coronary ischemia, trauma, and other related conditions. The infiltration of neutrophils is facilitated by the expression of adhesion molecules on the surface of endothelial cells. Particularly important are the selectin family of adhesion molecules at the onset of neutrophil-mediated injury. The aim of this study was to determine the role of selectin inhibition in the modulation of chemokine expression and Akt/MAPK signaling after liver I/R. In addition, we evaluated the optimal dose and time of administration of a small molecule selectin inhibitor, TBC-1269. Mice subjected to 90 min of partial (70-80%) hepatic ischemia followed by 3 h of reperfusion were divided into 15 groups (n = 4/group); sham, ischemic control, and 10, 20, and 40 mg/kg dose groups for the antiselectin molecule were studied at 3 times of drug administration: 1 h before reperfusion (but after ischemia), at the time of reperfusion, and at 15 min after reperfusion. The parameters measured after 3 h of reperfusion included liver function tests (ALT and AST), histopathology, and tissue myeloperoxidase (MPO). Chemokine expression (MIP-1alpha, MIP-1beta, MIP-2 and KC), Akt, MAPK (p44/p42), and RSK expressions were also measured in liver tissue by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. It was demonstrated that the small molecule multi-selectin inhibitor (TBC-1269) offered the most significant protection for the ischemic liver when given at 40 mg/kg at the time ofreperfusion. AST significantly differed between the control group and the group receiving 40 mg/kg at the time of reperfusion (p = .01). MPO levels in the liver tissue of the ischemic controls were significantly increased when compared to the levels of this enzyme in the TBC-1269 group at 40 mg/kg. Histological examination reflected the same results, with a significant difference (p = .02) between these same two groups. The chemokine profile also showed that the same treatment group had a downregulation of MIP-lalpha, MIP-1beta, MIP-2, and KC, as well as a lower expression of Akt, MAPK(p44/42), and RSK when compared to the control group. Thus, we demonstrated that the small molecule selectin inhibitor, TBC-1269, offered significant functional and structural protection of the ischemic liver when given at 40 mg/kg at the time of reperfusion. Lower doses and different times of administration did not show as prominent a drug effect. This selectin inhibition modulated the expression of Akt, MAPK (p44/42), and RSK, as well as MIP-1alpha, MIP-1beta, MIP-2, and KC chemokines. These alterations in cellular signaling and chemokine expression represent potential mechanisms or pathways of inflammatory response in I/R.     

NO donor ameliorates ischemia-reperfusion injury of the rat liver with iNOS attenuation.

This study investigated the effect of a spontaneous nitric oxide (NO) donor, FK409 (FK), in a rat model of segmental hepatic ischemia. Rats were allocated to four experimental groups. Two of the groups underwent segmental hepatic ischemia of 60 min duration and received FK (0.4 mg/kg, iv) or vehicle alone before inducing ischemia and again 5 min before reperfusion. Sham-FK and sham groups were treated identically, but did not have vascular occlusion. Serum aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase (LDH) were measured, and the livers were examined for histological evidence of injury, polymorphonuclear neutrophil (PMN) infiltration, and immunohistochemical expression of inducible NO synthase (iNOS) before and 6 h after reperfusion. AST, ALT, and LDH levels were significantly (p < .05) reduced 6 h after reperfusion in the FK-treated group compared with the vehicle-treated control group. FK treatment also reduced the degree of hepatic damage apparent on histopathology and reduced PMN infiltration and iNOS expression. Thus, FK treatment is protective against hepatic ischemia reperfusion injury and attenuates neutrophil infiltration and iNOS expression.     

Attenuation of liver and lung injury after hepatic ischemia and reperfusion by a cytokine-suppressive agent, FR167653

BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury is an important clinical problem and leads to the release of the proinflammatory cytokines, TNF-alpha and IL-1. These cytokines play important roles in the induction of polymorphonuclear neutrophil (PMN) activation and infiltration, and induce not only localized hepatic injury but also remote organ injury, especially pulmonary injury. Using a total hepatic ischemia model in rats, we tested our hypothesis that suppression of TNF-alpha and IL-1 by FR167653 ameliorates I/R injury in the liver and lung. METHODS: Male Wistar rats, weighing 240-280 g, were divided into 3 groups, an FR group, a control group and a sham group. In the FR group, FR167653 (1 mg/kg/h) was administered continuously to the animals for 30 min prior to the onset of ischemia and for 2 h after reperfusion. The control group received normal saline. A porto-systemic shunt was placed between the cecal branch of the portal vein and the jugular vein, and total hepatic ischemia was produced for 90 min. The sham group was treated with placement of the porto-systemic shunt only. The 1-week survival rate, liver enzyme activity, hepatic tissue blood flow (HTBF), cytokine mRNA expression, myeloperoxidase (MPO) activity and histological results were studied. RESULTS: The 1-week survival rate and HTBF were significantly higher in the FR group than in the control group. Serum AST, ALT, and LDH levels were significantly lower in the FR group at 30 min, 1 h and 3 h after reperfusion. MPO levels in liver and lung tissue were also significantly lower in the FR group. The expression of IL-1beta mRNA remarkably decreased up to 6 h after reperfusion in the FR group. CONCLUSIONS: We concluded that the inflammatory cytokines, IL-1beta, play important roles in hepatic I/R injury. FR167653 might ameliorate I/R injury and be useful in liver surgery with ischemia.     

Urinary trypsin inhibitor reduces C-X-C chemokine production in rat liver ischemia/reperfusion

BACKGROUND AND AIM: Protease inhibitors attenuate ischemia/reperfusion injury. However, the underlying mechanisms by which protease inhibitors prevent reperfusion injury remain obscure. Neutrophils play an important role in reperfusion injury. We studied the effects of urinary trypsin inhibitor (UTI) on production of the C-X-C chemokine, cytokine-induced neutrophil chemoattractant (CINC), by Kupffer cells during ischemia/reperfusion of the liver. METHODS: Liver ischemia was induced in rats by occlusion of the portal vein for 30 min. UTI (50,000 U/kg) was injected intravenously 5 min before vascular clamping. Serum CINC concentrations were measured by enzyme-linked immunosorbent assay. Levels of CINC mRNA in the liver were determined by Northern blot analysis. We also examined the inhibitory effects of UTI on in vitro CINC production by peritoneal macrophages in response to neutrophil elastase (NE). RESULTS: Serum CINC concentrations increased and peaked 6 h after reperfusion. However, pretreatment of animals with UTI blunted this increase in CINC and significantly reduced CINC mRNA levels in the liver after ischemia/reperfusion. UTI also decreased neutrophil accumulation in the liver 24 h after reperfusion. In vitro CINC production by Kupffer cells from rats pretreated with UTI 3 h after ischemia/reperfusion was significantly decreased compared to those from untreated animals. UTI reduced NE activity in vitro in a dose-dependent manner, and UTI significantly reduced in vitro CINC production by peritoneal macrophages stimulated with NE. CONCLUSION: UTI reduces the production of CINC by Kupffer cells stimulated with NE, attenuating ischemia/reperfusion injury of the liver.     

Neutrophil depletion and chemokine response after liver ischemia and reperfusion.

Neutrophils play a major role in the hepatic microvasculature following liver ischemia and reperfusion (I/R). Leukocyte cytokine chemoattractants (chemokines) are produced by neutrophils and cause neutrophil activation in I/R injury. We examined the role of neutrophils in the production of chemokines in the liver and lung inflammatory response following liver I/R. C57BL/6 mice were subjected to partial liver ischemia for 90 min. Four groups of animals were included: sham group, sham group with neutrophil depletion, ischemic control group, and ischemic control with neutrophil depletion. We evaluated at 3 h liver injury measurements, serum macrophage inflammatory protein-2 (MIP-2) and macrophage inflammatory protein-1 alpha (MIP-1alpha) chemokines, liver and lung tissue myeloperoxidase (MPO), and liver and lung histology. Statistical analysis included analysis of variance (ANOVA), and Student-Newman-Keuls and Kruskal-Wallis multiple comparison Z-value tests. Ischemic controls showed a significant increase in liver enzyme levels along with statistically significant higher liver and lung MPO activity values than the rest of the other groups (p < .05). MIP-2 values were higher in the ischemic control group when compared to the ischemic neutrophil depleted group. MIP-1alpha levels showed opposite results, being significantly lower (p < .05) in the ischemic control as compared to the neutrophil-depleted group. Improved liver and lung histopathological features were observed in the ischemic neutrophil depleted group when compared to the ischemic control group. Our study confirmed the key role of neutrophils in liver I/R injury and appeared to suggest some relationship between neutrophils and the production of certain chemokines, such as MIP-1alpha, which had an inverse relationship in the absence of neutrophils. Further studies will confirm the validity of these preliminary observations.     

Graft tumor necrosis factor receptor-1 protects after mouse liver transplantation whereas host tumor necrosis factor receptor-1 promotes injury

BACKGROUND: Tumor necrosis factor (TNF)-alpha is a cytokine with pleiotropic effects on the liver. The predominant hepatic receptor for TNFalpha is TNF receptor-1 (TNFR1). TNFR1 mediates liver injury after ischemia/reperfusion but is also mitogenic during hepatic regeneration. This study investigated the role of graft and host TNFR1 in early graft injury after liver transplantation in mice. METHODS: Livers from TNFR1 deficient (TNFR1-/-) and wild type (WT) mice were transplanted into either TNFR1-/- or WT recipients in all four possible combinations after 12 hours of cold storage. After eight hours, alanine transferase (ALT), necrosis, TdT-mediated dUTP-digoxigenin nick-end labeling (TUNEL) staining, caspase-3 activation, and myeloperoxidase were determined. RESULTS: When TNFR1-/- livers were transplanted into either WT or TNFR1-/- recipients, ALT was twofold greater than when WT donor livers were used. Necrosis and TUNEL staining also increased twofold and sevenfold, respectively, after transplantation of TNFR1-/- donor livers compared to WT. By contrast, ALT and necrosis decreased when WT or TNFR1-/- livers were transplanted into TNFR1-/- hosts compared to WT, which was associated with decreased neutrophil infiltration. CONCLUSION: In conclusion, graft and recipient TNFR1 has opposing effects. Graft TNFR1 decreases graft injury, whereas recipient TNFR1 mediates an increase of injury associated with enhanced neutrophil infiltration. Cross-transplanting of knockout and wild-type livers provides a new means to investigate graft-host interactions during hepatic injury.     

Kupffer cell tumor necrosis factor-alpha production is suppressed during liver regeneration

Mammalian liver regeneration following resection invokes intrinsic hepatic responses which result in rapid tissue repair. The role of soluble immune cytokines in this phenomenon is not known. The capacity of Kupffer cells (KC) from regenerating liver to produce the potent cytokine TNF-alpha was evaluated. Twenty-four hours after 70% partial hepatectomy (PHx) or sham operation, Kupffer cells were harvested from collagenase-digested Wistar-Furth rat livers and purified (greater than 95% by phagocytosis) by adherence. Following overnight culture with or without the cyclooxygenase inhibitor indomethacin (10 microM), 5 x 10(5) KC were repleted with fresh media with or without 2.5 micrograms/ml lipopolysaccharide (LPS). Supernatant TNF-alpha activities (units/ml) were measured using the L929 fibroblast lysis assay. With LPS, sham KC TNF-alpha levels were significantly higher (P less than 0.001) than those for PHx KC. Indomethacin significantly increased PHx KC TNF-alpha levels, but did not affect those for sham KC, suggesting autoregulation by arachidonic acid cyclooxygenase metabolites following PHx. We conclude that KC TNF-alpha production is suppressed following PHx by a mechanism apparently regulated by eicosanoid metabolism. During the stress of hepatic regeneration, a coordinated limitation of excessive TNF-alpha responses by PHx liver KC may naturally protect the host.     

Interleukin-6 mediates lung injury following ischemic acute kidney injury or bilateral nephrectomy

Patients with acute kidney injury frequently have pulmonary complications. Similarly ischemic acute kidney injury or bilateral nephrectomy in rodents causes lung injury characterized by pulmonary edema, increased pulmonary capillary leak and interstitial leukocyte infiltration. Interleukin-6 is a pro-inflammatory cytokine that is increased in the serum of patients with acute kidney injury and predicts mortality. Here we found that lung neutrophil infiltration, myeloperoxidase activity, the neutrophil chemokines KC and MIP-2 and capillary leak all increased within 4 h following acute kidney injury in wild-type mice. These pathologic factors were reduced in interleukin-6-deficient mice following acute kidney injury or bilateral nephrectomy. The lungs of mutant mice had reduced KC but MIP-2 was similar to that of wild type mice. Wild-type mice, treated with an interleukin-6 inactivating antibody, had decreased lung myeloperoxidase activity and KC levels following acute kidney injury. Our study shows that interleukin-6 contributes to lung injury following acute kidney injury.     

Effects of selective cyclooxygenase inhibitors on ischemia/reperfusion-induced hepatic microcirculatory dysfunction in mice

We examined the effects of selective cyclooxygenase (COX) inhibition on hepatic warm ischemia/reperfusion (I/R) injury in mice. A selective COX-1 inhibitor, SC-560, selective COX-2 inhibitors, NS-398 and celecoxib, and indomethacin were administered 30 min before ischemia. Four hours after reperfusion, an in vivo microscopic study showed that I/R caused significant accumulation of leukocytes adhering to the hepatic microvessels and nonperfused sinusoids. Levels of plasma alanine transaminase (ALT) and tumor necrosis factor (TNF)-alpha also showed increases. SC-560, NS-398, celecoxib and indomethacin significantly reduced hepatic responses to I/R including microcirculatory dysfunction and release of ALT and TNF-alpha. Moreover, the effects of the thromboxane (TX) A(2) (TXA(2)) synthase inhibitor OKY-046 and the TXA(2) receptor antagonist S-1452 on hepatic responses to I/R exhibited results similar to those obtained with COX inhibitors. These results suggest that COX-1 and COX-2 contribute to I/R-induced hepatic microvascular and hepatocellular injury partly through TNF-alpha production, and that TXs derived from COX are partly responsible for I/R-induced liver injury.     

Role of cyclic nucleotides in ischemia and reperfusion injury of canine livers

BACKGROUND: In a series of canine liver ischemia experiments, we have shown that amelioration of hepatic injury is achievable by the inhibition of vasoconstriction, cytokine production, platelet aggregation, and neutrophil infiltration. Cyclic adenosine diphosphate (cAMP) was considered to be involved in most of these events. In our study, we tested our hypothesis that augmentation of endogenous cAMP by phosphodiesterase (PDE) 3 inhibitor, amrinone (AM), or adenylate cyclase stimulator, NKH477 (NKH), could attenuate ischemia and reperfusion injury of the liver. METHODS: Thirty-six beagle dogs were used. They were divided into group CT (untreated control), group AM, group NKH, and group CB (treated by both agents). AM or NKH were administered i.v. 1 hr before ischemia (group preAM and group preNKH) or 15 min before reperfusion (pos-AM and postNKH). Combination group animals were treated only before ischemia. Animal survival, hepatic tissue blood flow, liver enzymes, platelet counts, energy metabolism, hepatic cAMP and cyclic guanosine 3',5'-cyclic monophosphate levels, and histopathology were analyzed. RESULTS: Two-week animal survival was significantly improved by pre- or posttreatment with either agent. After reperfusion, hepatic tissue blood flow, liver enzyme release, platelet counts, energy metabolism, tissue cAMP levels, and histological architecture were also ameliorated markedly. Combination of both agents induced severe liver damage and lethal hypotension. AM treatment exhibited more protective effects than NKH, particularly when it was given before ischemia. Interestingly, not only cyclic guanosine 3',5'-cyclic monophosphate, were also restored at higher levels after reperfusion by preischemia treatment. CONCLUSIONS: Administration of amrinone or NKH477 maintained hepatic tissue concentrations of cyclic nucleotides, and attenuated ischemia and reperfusion injury of the liver. Thus, regulation of hepatic tissue cyclic nucleotides is an important alternative for prevention of hepatic damage in liver preservation and surgery.     

Glutathione protects the rat liver against reperfusion injury after prolonged warm ischemia

OBJECTIVE: To evaluate the potential of postischemic intravenous infusion of the endogenous antioxidant glutathione (GSH) to protect the liver from reperfusion injury following prolonged warm ischemia. BACKGROUND DATA: The release of reactive oxygen species (ROS) by activated Kupffer cells (KC) and leukocytes causes reperfusion injury of the liver after warm ischemia. Therefore, safe and cost-effective antioxidant strategies would appear a promising approach to prevent hepatic reperfusion injury during liver resection, but need to be developed. METHODS: Livers of male Lewis rats were subjected to 60, 90, or 120 minutes of normothermic ischemia. During a 120 minutes reperfusion period either GSH (50, 100 or 200 micromol/h/kg; n= 6-8) or saline (n= 8) was continuously administered via the jugular vein. RESULTS: Postischemic GSH treatment significantly prevented necrotic injury to hepatocytes as indicated by a 50-60% reduction of serum ALT and AST. After 1 hour of ischemia and 2 hours of reperfusion apoptotic hepatocytes were rare (0.50 +/- 0.10%; mean +/- SD) and not different in GSH-treated animals (0.65 +/- 0.20%). GSH (200 micromol GSH/h/kg) improved survival following 2 hours of ischemia (6 of 9 versus 3 of 9 rats; P < 0.05). Intravital fluorescence microscopy revealed a nearly complete restoration of sinusoidal blood flow. This was paralleled by a reduction of leukocyte adherence to sinusoids and postsinusoidal venules. Intravenous GSH administration resulted in a 10- to 40-fold increase of plasma GSH levels, whereas intracellular GSH contents were unaffected. Plasma concentrations of oxidized glutathione (GSSG) increased up to 5-fold in GSH-treated animals suggesting counteraction of the vascular oxidant stress produced by activated KC. CONCLUSIONS: Intravenous GSH administration during reperfusion of ischemic livers prevents reperfusion injury in rats. Because GSH is well tolerable also in man, this novel approach could be introduced to human liver surgery.     

Role of leukotrienes on hepatic ischemia/reperfusion injury in rats

BACKGROUND: Leukotrienes (LT), composed of cysteinyl LT (cLT; LTC(4), LTD(4), and LTE(4)) and LTB(4), are potent lipid mediators enhancing the vascular permeability and recruitment of neutrophils, which are common features of hepatic ischemia/reperfusion (I/R) injury. The aim of this study was to investigate whether LT can mediate the liver and lung injuries following hepatic I/R. MATERIALS AND METHODS: Sprague-Dawley rats were subjected to 90 min of partial hepatic ischemia followed by 3, 12, and 24 h of reperfusion. In the hepatic and pulmonary tissues, LT content and the mRNA expression of LT-synthesis enzymes, 5-lypoxygenase (5-LO), LTC(4) synthase (LTC(4)-S), and LTA(4) hydrolase (LTA(4)-H) were measured. Tissue injuries were assessed by plasma ALT, histological examination, and wet-to-dry tissue weight ratios. RESULTS: The cLT content in the hepatic tissue after 12 and 24 h reperfusion was increased 4- to 5-fold compared to controls and this was accompanied by the enhancement of hepatic edema and plasma ALT elevation. There were no significant changes in the mRNA expression of LT-synthesis enzymes in both tissues. LTB(4) levels were not increased despite a significant neutrophil infiltration in both tissues. CONCLUSIONS: These data suggest that cLT are generated in the liver during the reperfusion period and may contribute to the development of hepatic edema and exert cytotoxicity. Factors other than LTB(4) may contribute to neutrophil infiltration.