Postischemic Liver Research Articles

The delay of rearterialization after initial portal reperfusion in living donor liver transplantation significantly determines the development of microvascular graft dysfunction

Background/Aims Graft reperfusion in liver transplantation is usually performed by initial portal reperfusion (IPR) and delayed rearterialization. Its influence on graft microcirculation is unknown. This study aimed to assess sinusoidal perfusion in dependence to this reperfusion technique during human living-donor liver transplantation. Methods Hepatic microcirculation was measured both in the donor and the recipient (n=14) by using the orthogonal polarization spectral imaging technique. By using initial portal reperfusion, the mean time interval between portal venous and hepatic arterial reperfusion was 27.7±13.3 min. Results Hepatic nutritive perfusion, as given by the functional sinusoidal density and the volumetric blood flow, was found significantly decreased during portal reperfusion when compared to baseline. Rearterialization resulted in hyperperfusion of individual sinusoids at a decreased density of the sinusoidal network. Interestingly, the time interval between portal venous and hepatic arterial reperfusion significantly correlated with the changes of the liver grafts' microcirculation. Conclusions The study indicates graft microcirculatory dysfunction as a major determinant of postischemic liver injury. Moreover, microvascular impairment was significantly influenced by the interval between portal venous and hepatic arterial reperfusion, which suggests the reinforcement of the pathomechanism of injury involving hypoxia and rapid graft rewarming due to initial portal reperfusion.

Superoxide and post-ischemic liver injury: potential therapeutic target for liver transplantation.

Cessation of blood flow to the liver is required during liver transplantation and resectional surgery. A growing body of experimental evidence suggests that restoration of blood flow to the ischemic liver initiates hepatocellular injury which may lead, in some cases, to severe liver injury and graft failure. A large number of studies have implicated reactive oxygen species as potential mediators of post-ischemic tissue injury. Recent developments in genetic engineering as well as chemical modeling, have allowed for the production of novel free radical scavengers including mutated forms of superoxide dismutase (SOD) and low molecular weight SOD mimics with extended circulating half-lives and/or significant membrane permeability's. Application of these newly developed free radical scavengers show promising results in animal models of liver I/R and may become powerful tools in the treatment of post-ischemic liver injury that occurs in liver transplantation.

Cytokine Cascades and the Hepatic Inflammatory Response to Ischemia and Reperfusion

Ischemia/reperfusion injury of the liver has two distinct phases that contribute to hepatocyte damage. The acute phase is characterized by Kupffer cell production of reactive oxygen species, which results in moderate hepatocyte injury. The later phase includes an intricate cascade of inflammatory events culminating in neutrophil infiltration of the postischemic liver. Accumulated neutrophils cause substantial injury to hepatocytes through their release of oxidants and proteases. This review discusses the mechanisms by which this inflammatory response is initiated, propagated, and regulated and the impact of these pathways on neutrophil-dependent injury to the liver.

Platelet–endothelial cell interactions during hepatic ischemia–reperfusion in vivo: a systematic analysis

Platelets have been implicated in the pathophysiology of ischemia–reperfusion (I/R) injury. This study was designed to analyze platelet–endothelial cell interactions after I/R of the liver in vivo in dependence of the duration of ischemia and reperfusion. In C57BL/6 mice, a warm (37°C) lobar hepatic ischemia was induced for 30, 60, or 90 min. Platelets were isolated from syngeneic donors, labeled ex vivo by rhodamine-6G, and infused intraarterially. Platelet–endothelial cell interactions and sinusoidal perfusion were analyzed using intravital fluorescence microscopy after 20–240 min of reperfusion. Hepatic I/R induced platelet rolling and adhesion in terminal arterioles and postsinusoidal venules as well as platelet accumulation in sinusoids already after 20 min of reperfusion. The number of platelets interacting with the endothelium was significantly increased as ischemia time was prolonged from 30 min up to 60 and 90 min. Postischemic platelet adhesion was associated with an increase in thrombin activity and a loss of platelets from the systemic circulation. Moreover, I/R-induced platelet adhesion was linearly correlated with an impairment of sinusoidal perfusion. The prolongation of reperfusion time up to 4 h did not further enhance platelet–endothelial cell interactions. These in vivo results indicate that hepatic I/R induces platelet–endothelial cell interactions in arterioles, sinusoids, and venules already during early reperfusion. The extent of these interactions is dependent on the duration of ischemia, but not on reperfusion time. Adherent platelets seem to participate in the development of sinusoidal perfusion failure in the postischemic liver.

Hepatic ischemia-reperfusion syndrome after partial liver resection (LR): Hepatic venous oxygen saturation, enzyme pattern, reduced and oxidized glutathione, procalcitonin and interleukin-6

The hepatic ischemia-reperfusion syndrome was investigated in 28 patients undergoing elective partial liver resection with intraoperative occlusion of hepatic inflow (Pringle maneuver) using the technique of liver vein catheterization. Hepatic venous oxygen saturation (ShvO2) was monitored continuously up to 24 hours after surgery. Aspartate aminotransferase, glutamate dehydrogenase, gamma-glutamyl transpeptidase, pseudocholinesterase, alpha-glutathione S-transferase, reduced and oxidized glutathione, procalcitonine, and interleukin-6 were serially measured both before and after Pringle maneuver during the resection and postoperatively in arterial and/or hepatic venous blood. ShvO2 measurement demonstrated that peri- and postoperative management was suitable to maintain an optimal hepatic oxygen supply. As expected, we were able to demonstrate a typical enzyme pattern of postischemic liver injury. There was a distinct decrease of reduced glutathione levels both in arterial and hepatic venous plasma after LR accompanied by a strong increase in oxidized glutathione concentration during the phase of reperfusion. We observed increases in procalcitonin and interleukin-6 levels both in arterial and hepatic venous blood after declamping. Our data support the view that liver resection in man under conditions of inflow occlusion resulted in ischemic lesion of the liver (loss of glutathione synthesizing capacity with disturbance of protection against oxidative stress) and an additional impairment during reperfusion (liberation of reactive oxygen species, local and systemic inflammation reaction with cytokine production). Additionally, we found some evidence for the assumption that the liver has an export function for reduced glutathione into plasma in man.

653 Relationship among complement activation, superoxide generation and cytokine expression in post-ischemic liver injury

653 Relationship among complement activation, superoxide generation and cytokine expression in post-ischemic liver injury

Cytokine Cascades and the Hepatic Inflammatory Response to Ischemia and Reperfusion

Ischemia/reperfusion injury of the liver has two distinct phases that contribute to hepatocyte damage. The acute phase is characterized by Kupffer cell production of reactive oxygen species, which results in moderate hepatocyte injury. The later phase includes an intricate cascade of inflammatory events culminating in neutrophil infiltration of the postischemic liver. Accumulated neutrophils cause substantial injury to hepatocytes through their release of oxidants and proteases. This review discusses the mechanisms by which this inflammatory response is initiated, propagated, and regulated and the impact of these pathways on neutrophil-dependent injury to the liver.

P-Selectin Mediates Platelet-Endothelial Cell Interactions and Reperfusion Injury in the Mouse Liver In Vivo

Platelets are suggested to participate in the pathogenesis of hepatic ischemia-reperfusion (I/R) injury. This study was designed to analyze platelet-endothelial cell interactions in the postischemic mouse liver in vivo and to define the role of endothelial versus platelet P-selectin for these interactions. Platelet-endothelial cell interactions were quantitatively analyzed using intravital fluorescence microscopy after lobar hepatic I/R in C57BL/6 wild-type and P-selectin-deficient mice after infusion of ex vivo rhodamine-6G-labeled wild-type and P-selectin-deficient platelets. Reperfusion injury and apoptosis were assessed by established methods. In wild-type animals, hepatic I/R caused significantly enhanced platelet-endothelial cell interactions in terminal arterioles and postsinusoidal venules as well as platelet stagnation in sinusoids. Concomitantly, transaminase and caspase-3 activities were elevated and sinusoidal perfusion was impaired. In contrast, platelet-endothelial cell interactions were nearly absent in arterioles and venules of mice lacking endothelial P-selectin, irrespective of the presence of P-selectin on infused platelets, but still significantly elevated in sinusoids. Simultaneously, sinusoidal perfusion failure was ameliorated, and transaminase- and caspase-3 activities were significantly reduced in P-selectin-deficient mice as compared with wild-type animals. The present intravital microscopic study provides, for the first time, quantitative analyses of platelet-endothelial cell interactions in the postischemic hepatic microcirculation. Our in vivo data show that endothelial P-selectin is critical for postischemic platelet-endothelial cell interactions within hepatic presinusoidal arterioles and postsinusoidal venules. P-selectin deficiency prevents microvascular injury and apoptosis after warm hepatic I/R.

Regulation of postischemic liver injury following different durations of ischemia.

The objective of this study was to define the relationship among Kupffer cells, O(2)(-) production, and TNF-alpha expression in the pathophysiology of postischemic liver injury following short and long periods of ischemia. Using different forms of superoxide dismutase with varying circulating half-lives, a monoclonal antibody directed against mouse TNF-alpha, and NADPH oxidase-deficient mice, we found that 45 or 90 min of partial (70%) liver ischemia and 6 h of reperfusion (I/R) produced time-dependent increases in liver injury and TNF-alpha expression in the absence of neutrophil infiltration. Furthermore, we observed that hepatocellular injury induced by short periods of ischemia were not dependent on formation of TNF-alpha but were dependent on Kupffer cells and NADPH oxidase-independent production of O(2)(-). However, liver injury induced by extended periods of ischemia appeared to require the presence of Kupffer cells, NADPH oxidase-derived O(2)(-), and TNF-alpha expression. We conclude that the sources for O(2)(-) formation and the relative importance of TNF-alpha in the pathophysiology of I/R-induced hepatocellular injury differ depending on the duration of ischemia.

Ischemic preconditioning attenuates the oxidant-dependent mechanisms of reperfusion cell damage and death in rat liver.

In an in vivo rat model of liver ischemia followed by reperfusion a consistent appearance of necrosis and activation of biochemical pathways of apoptosis was reproduced and monitored after 30 minutes reperfusion. Preconditioning by application of a short cycle of ischemia-reperfusion (10 minutes + 10 minutes) positively conditioned recovery of the organ at reperfusion, attenuating both necrotic and apoptotic events. Preconditioning at least halved cell oxidative damage occurring early at reperfusion, and as a major consequence, the increase of cytolysis and apoptosis occurring at reperfusion was about 50% less. The attenuation of both pathways of cell death by preconditioning appeared at least partly related to its modulate action on H(2)O(2) and 4-hydroxy-2,3-trans-nonenal production. The overall data point to a marked diminished oxidant generation and oxidative reactions as one major possible mechanism through which ischemic preconditioning exerts protection against necrotic and apoptotic insult to the postischemic liver.

Overexpression of redox factor-1 protects against postischemic liver injury by reducing oxidative stress and NF-κB activity

REPERFUSION of ischemic tissue results in the generation of reactive oxygen species (ROS). Parenchymal cells in the ischemic tissue contribute to oxidant stress through intracellular ROS generation early after reperfusion. In addition to direct tissue damage, ROS generated in postischemic tissue also functions as signaling intermediates that cause up-regulation of a variety of pro-inflammatory and pro-apoptotic molecules. Many of these substances are produced following activation of the redoxsensitive transcription factor NFB, an inflammatory reaction that compounds and perpetuates early reperfusion injury. For example, redox factor-1 (Ref-1), a bi-functional protein that is up-regulated in response to a host of stimuli that result in intracellular ROS generation, serves as an endonuclease in the base excision repair pathway, and also displays a reducing capacity that facilitates the DNAbinding activities of many redox-sensitive transcription factors including NFB. Both of these properties of Ref-1 may explain its role in cytoprotection against hypoxic and hyperoxic cell stresses in vitro. The present study examines the cytoprotective effects of Ref-1 in hepatic inflammation, apoptosis, and injury responses induced by ischemia-reperfusion in vivo.

Platelet adhesion mediated by fibrinogen-intercelllular adhesion molecule-1 binding induces tissue injury in the postischemic liver in vivo.

Platelets are thought to be involved in the induction of hepatic ischemia-reperfusion (I/R) injury. The mechanisms of platelet adhesion in the hepatic microvasculature and the role of platelets in the pathogenesis of I/R-induced liver damage in vivo remain unclear. In C57BL/6 mice, platelet- and leukocyte-endothelial cell interactions were quantitatively analyzed using intravital fluorescence microscopy in sham-operated animals, after warm lobar hepatic I/R (90/20 min) in wild-type and intercellular adhesion molecule (ICAM)-1-deficient mice, and after I/R in wild-type mice treated with an antifibrinogen antibody. Fibrinogen deposition on the endothelium was detected by intravital microscopy and by immunostaining. Reperfusion injury was assessed by measurement of liver enzyme and caspase-3 activities and of lipid peroxidation. Hepatic I/R induced fibrinogen deposition on hepatic endothelium, followed by a dramatic increase in the number of firmly adherent platelets in the liver microvasculature. Simultaneously, the number of adherent leukocytes in postsinusoidal venules and the aspartate aminotransferase/alanine aminotransferase and caspase-3 activities were elevated. Although ICAM-1 deficiency attenuated postischemic adherence of both platelets and leukocytes, the application of an antifibrinogen antibody selectively reduced the number of adherent platelets but did not influence leukocyte adhesion. The selective blockade of platelet adherence significantly prevented the postischemic increase in liver enzyme and caspase-3 activities. Furthermore, sinusoidal perfusion failure and lipid peroxidation were attenuated in the treated group. These in vivo data show that platelet adhesion mediated through fibrinogen deposition on ICAM-1 expressed on the endothelium of postischemic hepatic microvessels induces microvascular injury and hepatocellular apoptosis after I/R of the liver during early reperfusion.

Kupffer cell activation after no-flow ischemia versus hemorrhagic shock

Kupffer cell-derived oxidant stress is critical for reperfusion injury after no-flow ischemia. However, the importance of Kupffer cells as source of reactive oxygen formation is unclear in a hemorrhagic shock model. Therefore, we evaluated Kupffer cell activation after 60 or 120 min of hemorrhage and 90 min of resuscitation (HS/RS) in pentobarbital-anesthetized male Fischer rats. Plasma glutathione disulfide (GSSG) as indicator for a vascular oxidant stress showed no significant changes after HS/RS. Plasma ALT activities were only moderately increased (100–200 U/L). Kupffer cells isolated from postischemic livers did not generate more superoxide than cells from sham controls. In contrast, the 10-fold increase of plasma GSSG and the 9-fold higher spontaneous superoxide formation of Kupffer cells after 60 min of hepatic no-flow ischemia followed by 90 min of reperfusion demonstrated the activation of Kupffer cells in this experimental model. Plasma ALT activities (1930 ± 240 U/L) indicated severe liver injury. These results demonstrate a fundamental difference in the degree of Kupffer cell activation between the two models of warm hepatic ischemia. Our findings suggest that different therapeutic strategies are necessary to ameliorate the initial injury after low flow ischemia (hemorrhage) compared to cold (transplantation) or warm (Pringle maneuver) no-flow ischemia.

Transferrin receptor gene expression and transferrin-bound iron uptake are increased during postischemic rat liver reperfusion

Iron-catalyzed production of reactive oxygen species is a cause of liver injury after ischemia/reperfusion (I/R). The aim of the present study was to address the regulation of transferrin receptor (TfR), which mediates cellular iron uptake, during I/R. The molecular mechanisms controlling TfR gene expression in vivo during I/R of rat liver were investigated by molecular biology procedures. We also analyzed transferrin-bound iron uptake into surviving liver slices. Increased amounts of TfR protein and messenger RNA (mRNA) were found 2 to 6 hours after reestablishment of blood supply. RNA bandshift analysis showed that iron regulatory protein (IRP) activity was decreased in the first hours of reperfusion, thus indicating that IRP-mediated mRNA stabilization was not involved in early TfR upregulation. On the contrary, increased transcription of the TfR gene in isolated nuclei was observed during reperfusion; during the ischemic phase this was preceded by enhanced binding of hypoxia inducible factor (HIF-1) to a DNA sequence derived from the TfR promoter. TfR2 mRNA levels were also enhanced after reperfusion. The increased expression of TfR at the cell surface resulted in increased uptake of transferrin-bound-iron into surviving liver slices; however, iron was not incorporated into ferritin. In conclusion, HIF-1 mediated activation of TfR gene transcription and IRP-mediated increase of TfR mRNA stability ensure a steady induction of TfR, and hence higher iron uptake in reperfused rat liver. TfR-mediated entry of the metal into liver cells may represent a source of catalitically active iron, which may play a role in reperfusion damage. (H EPATOLOGY 2002;36:103-111.)

Molecular mechanisms of leukocyte recruitment in postischemic liver microcirculation.

Evidence shows that leukocyte recruitment into inflamed liver sinusoids does not require selectins, with one notable exception: ischemia-reperfusion (I/R). We used intravital microscopy to directly visualize the liver microcirculation during I/R and localized endotoxemia (liver superfused with lipopolysaccharide). General anti-selectin therapy (fucoidan) or anti-adhesion therapy with an antithrombin inhibitor (hirudin) was also used. Many neutrophils rolled and adhered in postsinusoidal vessels and sequestered in the sinusoids during I/R and local endotoxin superfusion. Although fucoidan blocked rolling in both forms of inflammation, leukocyte recruitment into sinusoids was only blocked in I/R. Adhesion was also inhibited in postischemic sinusoids with a second anti-adhesive agent (hirudin). Because liver I/R inevitably induces ischemia upstream in the intestine, anti-selectin therapy may prevent intestinal injury, which could prevent downstream liver inflammation. To test this hypothesis, we completely removed the intestine and rerouted blood flow from the superior mesenteric artery to the superior mesenteric vein. I/R was induced in the liver microcirculation, and many leukocytes rolled and adhered in postsinusoidal venules and adhered in sinusoids. Although fucoidan significantly reduced the rolling in postsinusoidal vessels, adhesion persisted in the sinusoids. Our data suggest that anti-adhesion therapy is effective in liver I/R in the sinusoids and postsinusoidal venules, perhaps in part due to its beneficial effect on the intestine.

Role of nitric oxide in liver ischemia and reperfusion injury.

The present study was designed to assess the role of endothelial cell and inducible nitric oxide synthase (eNOS, iNOS)-derived NO in ischemia/reperfusion (I/R)-induced pro-inflammatory cytokine expression and tissue injury in a murine model of hepatic I/R. Forty-five min of partial hepatic ischemia and 3 h of reperfusion resulted in a significant increase in liver injury as assessed by serum alanine aminotransferase and histopathology which occurred in the absence of neutrophil infiltration. Both iNOS and eNOS deficient mice exhibited enhanced liver injury when compared to their wild type (wt) controls again in the absence of neutrophil infiltration. Interestingly, message expression for both tumor necrosis factor-alpha (TNF-alpha) and interleukin 12 (IL-12) were enhanced in eNOS, but not iNOS-deficient mice at 1 h post-ischemia when compared to their wt controls. In addition, eNOS message expression appeared to be up-regulated between 1 and 3 h ofreperfusion in wt mice while iNOS deficient mice exhibited substantial increases at I but not 3 h. Taken together, these data demonstrate the ability of eNOS and iNOS to protect the post-ischemic liver, however their mechanisms of action may be very different.

Enhanced Post-Ischemic Liver Injury in iNOS-Deficient Mice: A Cautionary Note

The objective of this study was to assess the role of inducible nitric oxide synthase (iNOS) in ischemia- and reperfusion (I/R)-induced liver injury. We found that partial hepatic ischemia involving 70% of the liver resulted in a time-dependent increase in serum alanine aminotransferase (ALT) levels at 1–6 h following reperfusion. Liver injury at 1, 3, and 6 h post-ischemia was not due to the infiltration of neutrophils as assessed by tissue myeloperoxidase (MPO) activity and histopathology. iNOS-deficient mice subjected to the same duration of ischemia and reperfusion showed dramatic and significant increases in liver injury at 3 but not 6 h following reperfusion compared to their wild type controls. Paradoxically, iNOS mRNA expression was not detected in the livers of wild type mice at any point during the reperfusion period and pharmacological inhibition of iNOS using l-N6(iminoethyl)-lysine (l-NIL) did not exacerbate post-ischemic liver injury at any time post-reperfusion. These data suggest that iNOS deficiency produces unanticipated genetic alterations that renders these mice more sensitive to liver I/R-induced injury.

Endothelin B receptor–Mediated protection against anoxia-reoxygenation injury in perfused rat liver: Nitric oxide–Dependent and –Independent mechanisms

This study aimed to investigate the roles of endothelin (ET) receptors in biliary dysfunction and cell injury in postischemic livers. Rat livers perfused with oxygenated Krebs-Henseleit solution were exposed to reoxygenation following 20-minute hypoxia. The anoxic perfusion decreased bile output and reduced cyclic guanosine monophosphate (cGMP) contents, an index of nitric oxide (NO) generation. Upon reoxygenation, the decreased bile was not fully recovered, and the resistance increased biphasically: an early transient spike accompanied by an elevated release of ET-1 and a rise accompanied by a cGMP elevation in the later period. The initial vasoconstriction appeared to be mediated by both ETA and ETB receptors, as judged by inhibitory effects of their antagonists, BQ-485 and BQ–788, respectively, while the late elevation of the resistance was not attenuated by these reagents, but rather enhanced by the ETB blockade. The BQ-788 treatment attenuated the reoxygenation-induced cGMP elevation and induced bile acid–dependent choleresis. However, such a change upon the ETB blockade coincided with dissociation of a recovery of phospholipids and aggravation of cell injury. The BQ-788–elicited deterioration of reoxygenation-elicited changes was attenuated by NO supplement with S-nitroso-N-acetyl penicillamine. Nω-Nitro-L-arginine methyl ester, an NO synthase inhibitor, mimicked biliary changes elicited by the ETB blockade but without causing notable cell injury. Under these circumstances, coadministration of clotrimazole, an inhibitor of cytochrome P450 mono-oxygenases, elicited the injury comparable with that observed under the ETB blockade. These results suggest that ETB-mediated signaling limits excessive bile acid excretion and plays a protective role against reoxygenation injury through mechanisms involving both NO-dependent and -independent processes. (HEPATOLOGY 2001;33:894-901.)

Role of nitric oxide in regulating cytokine expression and tissue injury in postischemic liver injury

Role of nitric oxide in regulating cytokine expression and tissue injury in postischemic liver injury

Role of nitric oxide in regulating cytokine expression and tissue injury in postischemic liver injury

Role of nitric oxide in regulating cytokine expression and tissue injury in postischemic liver injury