Tacrolimus preconditioning of rat liver allografts impacts glutathione homeostasis and early reperfusion injury.

OBJECTIVE: To characterize the immunosuppressant tacrolimus as a protective antioxidant in rat liver transplantation. METHODS: Livers of male Lewis rats underwent 24 h of hypothermic preservation in UW solution and were rinsed with tacrolimus or placebo directly before transplantation. Markers of liver injury, such as enzymes and bile flow, were determined during a 2 h reperfusion period. Concentrations of reduced (GSH) and oxidized (GSSG) glutathione were analyzed in plasma, bile, and liver tissue for estimation of oxidant stress caused by reactive oxygen species (ROS). RESULTS: Administration of tacrolimus (10 ng/mL) resulted in decreased ALT plasma levels (1740 ± 1169 U/l versus 3691 ± 1144 U/l; P < 0.05) at 2 h of reperfusion. While endogenous intracellular GSH concentrations remained unchanged, GSSG, the oxidation product of GSH, was markedly decreased at 2 h of reperfusion in preconditioned livers (47.0 ± 10.4 nm/g versus 71.8 ± 30.6 nm/g; P < 0.05). Correspondingly, GSSG bile concentrations (0.19 ± 0.04 mM versus 0.13 ± 0.04 mM; P < 0.05) as well as plasma GSSG levels (2.4 ± 0.3 mM versus 1.4 ± 0.2 mM; P < 0.05) were significantly increased upon reperfusion. These findings suggest that tacrolimus impacts post-ischemic GSH metabolism when administered as a rinse solution for liver allografts through an unknown pathway. CONCLUSION: Hepatocellular injury following transplantation was significantly decreased by preconditioning with tacrolimus. One possible mechanism of action is the detoxification of ROS through the preservation of cytosolic and extracellular GSH/GSSG ratios.

Ischemic preconditioning-induced hyperperfusion correlates with hepatoprotection after liver resection.

AIM: To characterize the impact of the Pringle maneuver (PM) and ischemic preconditioning (IP) on total blood supply to the liver following hepatectomies. METHODS: Sixty one consecutive patients who underwent hepatic resection under inflow occlusion were randomized either to receive PM alone (n = 31) or IP (10 min of ischemia followed by 10 min of reperfusion) prior to PM (n = 30). Quantification of liver perfusion was measured by Doppler probes at the hepatic artery and portal vein at various time points after reperfusion of remnant livers. RESULTS: Occlusion times of 33 +/- 12 min (mean +/- SD) and 34 +/- 14 min and the extent of resected liver tissue (2.7 segments) were similar in both groups. In controls (PM), on reperfusion of liver remnants for 15 min, portal perfusion markedly decreased by 29% while there was a slight increase of 8% in the arterial blood flow. In contrast, following IP + PM the portal vein flow remained unchanged during reperfusion and a significantly increased arterial blood flow (+56% vs baseline) was observed. In accordance with a better postischemic blood supply of the liver, hepatocellular injury, as measured by alanine aminotransferase (ALT) levels on day 1 was considerably lower in group B compared to group A (247 +/- 210 U/I vs 550 +/- 650 U/I, P < 0.05). Additionally, ALT levels were significantly correlated to the hepatic artery inflow. CONCLUSION: IP prevents postischemic flow reduction of the portal vein and simultaneously increases arterial perfusion, suggesting that improved hepatic macrocirculation is a protective mechanism following hepatectomy.

Surgical treatment of primary biliary cirrhosis and primary sclerosing cholangitis.

Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are progressive cholestatic liver diseases of supposed auto-immune etiology. The clinical course is unpredictable and, in many patients, leads to end-stage liver disease or a poor quality of life. Conservative therapy only has a limited effect on the natural history, but orthotopic liver transplantation(OLT) offers a definitive therapeutic option. Retrospective analysis was performed for 38 patients with PBC and 17 patients with PSC who underwent OLT between January 1986 and June 2003 at our institution. Median followup after OLT was 72 mo.Cumulative survival at 5 yr post-OLT was 84% in the PBC group and 73% in the PSC group. Compared with OLT for other benign diseases, actuarial survival rates at 5 and 10 yr post-OLT were significantly better for patients with PBC, whereas there was no difference in survival after OLT for patients with PSC. Survival rate at 5 yr post-OLT was significantly increased for patients with PBC who had a Child-Pugh B liver cirrhosis (93%) compared with those who had Child-Pugh C cirrhosis (60%). Retransplantation rate was 18.2% (resulting from biliary complications in three cases). Surgical techniques had no effect on outcome after OLT in both groups. We concluded that liver transplantation represents a safe and beneficial therapy for patients with end-stage PBC. Cirrhotic patients with PSC also benefit from OLT, with an outcome comparable to that of liver cirrhosis of other etiologies.

Alpha-gluthathione S-transferase as an early marker of hepatic ischemia/reperfusion injury after liver resection.

Organ dysfunction following liver resection is one of the major postoperative complications of liver surgery. The Pringle maneuver is often applied during liver resection to minimize bleeding, which in turn complicates the postoperative course owing to liver ischemia and reperfusion. Routinely, hepatocellular damage is diagnosed by, for example, abnormal aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and the prothrombin time (PT). The cytosolic liver enzyme alpha-glutathione S-transferase (alpha-GST) has recently been shown to have good sensitivity for detecting hepatic injury after acetaminophen poisoning or liver transplantation, but its role in non-transplantation liver surgery has not been assessed. In this prospective randomized clinical study, the diagnostic role of plasma alpha-GST following warm ischemia and reperfusion is reported. A total of 75 patients who underwent liver resection were randomly assigned to three groups: (1) without Pringle (NPR); (2) with Pringle (PR); (3) with ischemic preconditioning by 10 minutes of ischemia and reperfusion each prior to the Pringle manuever (IPC). The major findings are as follows: (1) ALT, AST, and alpha-GST increased upon liver manipulation as early as prior to resection, with a rapid return of alpha-GST values to preoperative levels, whereas ALT and AST further increased on the first postoperative day. (2) In the PR group, alpha-GST, but not ALT and AST, was significantly elevated compared with that in the NPR group at 15 and 30 minutes and 2 hours after resection/reperfusion. In addition, only levels of alpha-GST significantly correlated with the Pringle duration. (3) The ischemia/reperfusion-induced early rise in alpha-GST was completely prevented by ischemic preconditioning. Moreover, only alpha-GST concentrations (> 490 microg L(-1)) determined early after resection (2 hours) predicted postoperative liver dysfunction (24 hours PT < 60%) with a positive predictive value of 74% and a negative predictive value of 76%. Thus alpha-GST seems to be a sensitive, predictive marker of ischemia/reperfusion-induced hepatocellular injury and postoperative liver dysfunction.

Intravenous administration of glutathione protects parenchymal and non-parenchymal liver cells against reperfusion injury following rat liver transplantation.

AIM: To investigated the effects of intravenous administration of the antioxidant glutathione (GSH) on reperfusion injury following liver transplantation. METHODS: Livers of male Lewis rats were transplanted after 24 h of hypothermic preservation in University of Wisconsin solution in a syngeneic setting. During a 2-h reperfusion period either saline (controls, n=8) or GSH (50 or 100 micromol/(h/kg), n=5 each) was continuously administered via the jugular vein. RESULTS: Two hours after starting reperfusion plasma ALT increased to 1457+/-281 U/L (mean+/-SE) in controls but to only 908+/-187 U/L (P<0.05) in animals treated with 100 microGSH/(h/kg). No protection was conveyed by 50 micromol GSH(h/kg). Cytoprotection was confirmed by morphological findings on electron microscopy: GSH treatment prevented detachment of sinusoidal endothelial cells (SEC) as well as loss of microvilli and mitochondrial swelling of hepatocytes. Accordingly, postischemic bile flow increased 2-fold. Intravital fluorescence microscopy revealed a nearly complete restoration of sinusoidal blood flow and a significant reduction of leukocyte adherence to sinusoids and postsinusoidal venules. Following infusion of 50 micromol and 100 micromol GSH/(h/kg), plasma GSH increased to 65+/-7 mol/L and 97+/-18 mol/L, but to only 20+/-3 mol/L in untreated recipients. Furthermore, plasma glutathione disulfide (GSSG) increased to 7.5+/-1.0 mol/L in animals treated with 100 micro(h/kg) GSH but did not raise levels of untreated controls (1.8+/-0.5 mol/L) following infusion of 50 microGSH/(h/kg) (2.2+/-0.2 mol/L). CONCLUSION: Plasma GSH levels above a critical level may act as a "sink" for ROS produced in the hepatic vasculature during reperfusion of liver grafts. Therefore, GSH can be considered a candidate antioxidant for the prevention of reperfusion injury after liver transplantation, in particular since it has a low toxicity in humans.

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.

Induction of cellular resistance against Kupffer cell-derived oxidant stress: a novel concept of hepatoprotection by ischemic preconditioning.

Ischemic preconditioning (IP) triggers protection of the liver from prolonged subsequent ischemia. However, the underlying protective mechanisms are largely unknown. We investigated whether and how IP protects the liver against reperfusion injury caused by Kupffer cell (KC)-derived oxidants. IP before 90 minutes of warm ischemia of rat livers in vivo significantly reduced serum alanine aminotransferase (AST) levels and leukocyte adherence to sinusoids and postsinusoidal venules during reperfusion. This protective effect was mimicked by postischemic intravenous infusion of glutathione (GSH), an antioxidative strategy against KC-derived H(2)O(2). Interestingly, no additional protection was achieved by infusion of GSH to preconditioned animals. These findings and several additional experiments strongly suggest IP mediated antioxidative effects: IP prevented oxidant cell injury in isolated perfused rat livers after selective KC activation by zymosan. Moreover, IP prevented cell injury and pertubations of the intracellular GSH/GSSG redox system caused by direct infusion of H(2)O(2) (0.5 mmol/L). IP-mediated resistance against H(2)O(2) could neither be blocked by the adenosine A2a antagonist DMPX nor mimicked by A2a agonist CGS21680. In contrast, H(2)O(2) resistance was abolished by the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580, but induced when p38 MAPK was directly activated by anisomycin. In conclusion, we propose a novel concept of hepatoprotection by IP: protection of liver cells by enhancing their resistance against KC-derived H(2)O(2). Activation of p38 MAPK and preservation of the intracellular GSH/oxidized glutathione (GSSG) redox system, but not adenosine A2a receptor stimulation, seems to be pivotal for the development of H(2)O(2) resistance in preconditioned livers.