Splenic artery ligation: A protection against hepatic ischemia/reperfusion injury in partially hepatectomized rats

Aim: In liver resection, the temporary occlusion of the hepatoduodenal ligament (Pringle maneuver) is often used. However, the maneuver causes ischemia/reperfusion (I/R) injury in the remnant liver. Heme oxygenase (HO)‐1 has a cytoprotective role against this injury. Our aim is to investigate whether splenic artery ligation induces HO‐1 expression in the liver and ameliorates the hepatic I/R injury in partially hepatectomized rats. Methods: Rats underwent splenic artery ligation by occluding the main splenic artery. Two days later, the total hepatic ischemia (Pringle maneuver) was conducted, and then a two‐thirds partial hepatectomy (PH) was performed just before the start of reperfusion. HO inhibitor was twice injected s.c. at 3 and 16 h before the Pringle maneuver. HO‐1 levels were determined by western blotting. Liver injury was biochemically assessed. Results: In normal rats, HO‐1 was highly expressed in the spleen, but not in the liver. Splenic artery ligation induced HO‐1 in the livers. When rats underwent 20 and 30 min of Pringle maneuver/PH, survival rates were 28% and 8%, respectively. Splenic artery ligation significantly improved both the survival rates: 73% and 56%, respectively. Under these conditions, administration of HO‐1 inhibitor at least partly negated the efficacy of splenic artery ligation. Splenic artery ligation also increased the recovery rate of the remnant liver mass and platelet counts in Pringle maneuver/PH‐treated rats. Conclusion: Splenic artery ligation was significantly effective on the hepatic I/R injury in partially hepatectomized rats. Induction of HO‐1 may be at least partly involved in the improvement of this injury.     

Effects of prior splenectomy on remnant liver after partial hepatectomy with Pringle maneuver in rats.

BACKGROUND/AIMS: In the case of the liver resection, the temporary occlusion of the hepatoduodenal ligament (Pringle maneuver) is often used. However, the maneuver causes hepatic ischemia/reperfusion (I/R) injury that strongly affects the recovery of patients. The present study investigated the effects of prior splenectomy on the remnant liver in partial hepatectomized rat with Pringle maneuver. METHODS: Pringle maneuver was conducted just before a two-thirds partial hepatectomy. Efficacy of splenectomy was assessed by survival rate, serum alanine aminotransferase (ALT), neutrophil infiltration into liver, recovery of remnant liver weight, and liver proliferating cell nuclear antigen (PCNA) levels. Ischemic preconditioning was performed as follows; 10 min of total hepatic ischemia followed by 10 min of reperfusion. RESULTS: In partial hepatectomized rats with 30 min of Pringle maneuver, seven out of 12 rats died within 3 days. On the other hand, when splenectomy was performed on 3 days before the maneuver, only one out of 12 rats died. When prior splenectomy was performed on eight and 18 days before the Pringle maneuver, respectively, similar efficacy was observed. In addition, prior splenectomy on 3 days before the maneuver showed that serum ALT activity, neutrophil infiltration, recovery of remnant liver weight, and PCNA levels in partial hepatectomized rats with Pringle maneuver were also ameliorated as compared with those of control rats without splenectomy. When effects of prior splenectomy were compared with those of ischemic preconditioning in these situations, efficacy of prior splenectomy was comparable with that of the ischemic preconditioning. CONCLUSIONS: Prior splenectomy ameliorated the I/R injury in the remnant liver after partial hepatectomy with Pringle maneuver. Effects of prior splenectomy may influence the liver for long duration, because splenectomy on 18 days before the maneuver still exerts effective action.     

Splenic artery ligation improves remnant liver function in partially hepatectomized rats with ischemia/reperfusion injury

Background: In liver resection, the temporary occlusion of the hepatoduodenal ligament (Pringle maneuver) is often used. However, the maneuver causes severe ischemia/reperfusion injury in the remnant liver. Our aim was to investigate the effects of splenic artery ligation on the liver function in partially hepatectomized rat with the Pringle maneuver. Methods: The Pringle maneuver was conducted for 30 min just before a two‐thirds partial hepatectomy. Splenic artery ligation was performed before the Pringle maneuver. The efficacy of splenic artery ligation was assessed by survival, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), recovery of remnant liver weight, and portal pressure. Results: On day 3, animal survival was four rats of 12 in partially hepatectomized rats with the Pringle maneuver and 10 rats of 12 in the splenic artery ligation‐treated partially hepatectomized rats with the Pringle maneuver. A two‐thirds partial hepatectomy alone or splenic artery ligation itself did not show any effects on the survival. Compared with partially hepatectomized rats with the Pringle maneuver, splenic artery‐ligated animals had lower serum AST and ALT levels, and higher recovery of remnant liver weight. Splenic artery ligation significantly reduced the portal pressure and also decreased the fatality in excessively hepatectomized rats. Conclusions: Splenic artery ligation ameliorated the remnant liver function in partially hepatectomized rats with the Pringle maneuver and excessively hepatectomized rats. The amelioration may be mediated at least by decreasing portal pressure.     

Impaired liver regeneration following partial hepatectomy using the Pringle maneuver: Protective effect of mesna

Background and Aim: We investigated the role of the prophylactic administration of the antioxidant 2‐mercaptoethane sulfonate (mesna) on the hepatocyte‐regenerating capacity following partial hepatectomy (PH) with concurrent Pringle maneuver. Methods: Wistar rats were subjected to PH (70% hepatectomy), 30 min Pringle maneuver, PH plus Pringle with or without mesna pretreatment (400 mg/kg, per os, 3 h before Pringle), or sham operation. At 24 h, 48 h, 72 h, and 1 week after operation, relative liver weight, hepatocyte mitotic activity (mitotic index), the histopathological score and serum aspartate aminotransferase, and alanine aminotransferase concentrations were assessed. At 1 h after operation, oxidative stress markers (glutathione to glutathione disulfide ratio, malondialdehyde concentration, and superoxide dismutase activity) and nuclear factor‐κB (NF‐κB) activity were assessed. Results: Hepatectomy stimulated the regenerating process and induced mild oxidative stress and the activation of NF‐κB in hepatocytes, while causing tissue injury in the remnant liver. When PH was performed under Pringle maneuver, hepatocyte mitotic activity was substantially suppressed, although Pringle alone initiated a delayed regenerating response. Furthermore, Pringle maneuver deteriorated oxidative stress markers, markedly increased NF‐κB activity, and aggravated tissue injury, as compared to hepatectomy alone. Mesna pretreatment prevented the Pringle‐induced antimitotic effect and the induction of oxidative stress, inhibited the activation of NF‐κB, while attenuating liver injury after PH under Pringle. Conclusion: The excessive activation of NF‐κB is related to the suppression of hepatocyte‐regenerating activity following PH with concurrent liver ischemia. Mesna pretreatment protects the liver against the Pringle‐induced antimitotic effect after PH via the prevention of oxidative stress and the inhibition of NF‐κB activation.     

Splenic artery ligation ameliorates hepatic ischemia and reperfusion injury in rats.

BACKGROUND/AIMS: Hepatic injury caused by ischemia/reperfusion (I/R) is a key clinical problem associated with liver transplantation and liver surgery. The spleen is involved in hepatic I/R injury. In this study, we examined the effects of splenic artery ligation on hepatic I/R injury. METHODS: Splenic artery ligation was performed 7 days, 3 days, or just before the hepatic ischemia. Hepatic ischemia was conducted by occluding the blood vessels to the median and left lateral lobes with an atraumatic vascular clamp. Hepatic I/R injury was induced by 45 min of ischemia followed by 120 min of reperfusion. RESULTS: When splenic artery ligation was performed at 3 days or just before the ischemia, serum aspartate transaminase and alanine transaminase activities, as markers for hepatic injury, decreased as compared with the rats with I/R alone. Splenic artery ligation also reduced the myeloperoxidase activity, an enzyme present in neutrophils, and the expression of interleukin-6 mRNA, a proinflammatory cytokine, in rat livers with I/R. Efficacy of splenic artery ligation on hepatic I/R injury was also confirmed by histology. On the other hand, when splenic artery ligation was conducted 7 days before the ischemia, efficacy of splenic artery ligation was disappeared. CONCLUSIONS: Splenic artery ligation ameliorates hepatic I/R injury in rats. These results strongly suggest the clinical usefulness of this surgical procedure to protect the liver against I/R injury.     

Comparison of continuous versus intermittent hepatic pedicle clamping in an experimental model.

BACKGROUND/AIMS: The tolerance of the liver to ischemia obtained with intermittent clamping of the hepatic pedicle or continuous Pringle maneuver was tested. METHODOLOGY: Ninety rats were divided into three groups undergoing total duration of clamping ischemia of 60, 90, and 120 min. Each group of rats were subdivided to receive continuous Pringle maneuver, 30-min or 15-min intermittent clamping. The clamp release time between the periods of liver ischemia was 5 min. Survival at 7 days and postoperative changes of liver function (transaminase enzymes, bilirubin, and adenosine-5'-triphosphate levels (hepatocellular damage index) were recorded. RESULTS: Intermittent clamping of the hepatic pedicle was better tolerated than the continuous clamping method. With continuous clamping the rat survival rates inversely correlated with the duration of ischemia. Survival rates at 15-min and 30-min intermittent ischemia groups were significantly higher than in the continuous clamping group. CONCLUSIONS: This data suggest that when the Pringle maneuver is adopted, it should be applied intermittently rather than continuously.     

Japanese herbal medicine, Saiko-keishi-to, prevents gutischemia／reperfusion-induced liver injurv in rats via nitric oxide

AIM: To determine whether Saiko-keishi-to (TJ-10), a Japanese herbal medicine, could protect liver injury induced by gut ischemia/reperfusion (I/R), and to investigate the role of NO. METHODS: Male Wistar rats were exposed to 30-min gut ischemia followed by 60 min of reperfusion. Intravital microscopy was used to monitor leukocyte recruitment. Plasma tumor necrosis factor (TNF) levels and alanine aminotransferase (ALT) activities were measured. TJ-10 1 g/(kg.d) was intragastrically administered to rats for 7 d. A NO synthase inhibitor was administered. RESULTS: In control rats, gut I/R elicited increases in the number of stationary leukocytes, and plasma TNF levels and ALT activities were mitigated by pretreatment with TJ-10. Pretreatment with the NO synthase inhibitor diminished the protective effects of TJ-10 on leukostasis in the liver, and the increase of plasma TNF levels and ALT activities. Pretreatment with TJ-10 increased plasma nitrite/nitrate levels. CONCLUSION: TJ-10 attenuates the gut I/R-induced hepatic microvascular dysfunction and sequential hepatocellular injury via enhancement of NO production.     

Successful intermittent application of the Pringle maneuver for 30 minutes during human hepatectomy: a clinical randomized study with use of a protease inhibitor

BACKGROUND/AIMS: There is experimental evidence that the liver can safely tolerate a cycle involving application of the Pringle maneuver for 30 minutes each time. METHODOLOGY: One hundred and twenty patients who underwent elective hepatectomy were randomly assigned to two groups of intermittent occlusion (30-min or 15-min Pringle group, n=60 each). A synthetic protease inhibitor (gabexate mesilate, GM, 2 mg/kg/h) was administered to pharmacologically alleviate visceral congestion, accompanied by hepatic pedicle clamping. Intraoperative data, liver function parameters and portal plasma levels of the inflammatory cytokine, interleukin (IL)-6 (a marker reflecting the status of visceral congestion), were examined as well as the postoperative course. Additionally, ten more patients randomly underwent right hepatectomy without GM, in order to clarify the influence of this agent on the present outcomes. RESULTS: The two groups of patients were comparable in terms of preoperative assessments, hepatic inflow occlusion time, extent of resection and background liver conditions. The 30-min Pringle group showed less blood loss during surgery (p=0.02) with a tendency for better postoperative mortality and morbidity. The postoperative liver functions were similar between the two groups. The portal plasma levels of IL-6 during pedicle clamping did not differ significantly between the two groups. When GM was not used, the 30-min intermittent Pringle maneuver induced a two-fold rise in serum transaminase levels on day 1 compared with the 15-min group. CONCLUSIONS: Our study indicates that intermittent application of the Pringle maneuver for 30 minutes each time can be accomplished effectively and safely for human hepatectomy, when combined with use of a protease inhibitor.     

Dihydrolipoyl histidinate zinc complex, a new antioxidant, attenuates hepatic ischemia-reperfusion injury in rats

Background and Aims: Ischemia/reperfusion (I/R) injury is characterized by significant oxidative stress, which induces characteristic changes in the antioxidant system and organ injury leading to significant morbidity and mortality. The aim of this study was to evaluate the protective effect of dihydrolipoyl histidinate zinc complex (DHLHZn) on oxidative damage after severe hepatic I/R injury. Methods: Thirty male Wistar rats were subjected to 45 min of hepatic ischemia by clamping of the hepatic artery and portal vein, followed by a 6‐h reperfusion period. DHLHZn (10 mg/kg) (I/R + DHLHZn group) or saline (I/R group) was administered intraperitoneally twice, 30 min before ischemia and at the beginning of the reperfusion. Sham‐operated animals (sham group) received equal amounts of saline. The rats were killed at the end of the reperfusion period. Serum levels of aspartate aminotransferase and alanine aminotransferase were determined, and histological examination and oxidative stress were evaluated in liver tissues. In addition, antimycin A‐stimulated RAW264.7 cells (murine macrophage‐like cells) were treated with DHLHZn to estimate its antioxidant effect. Results: Serum aspartate aminotransferase and alanine aminotransferase levels were increased in the I/R group, but these increases were significantly inhibited in the I/R + DHLHZn group. Similarly, liver tissue damage observed in the I/R group was attenuated in the I/R + DHLHZn group. Cells treated in vitro with both DHLHZn and antimycin A showed reduced reactive oxygen species activity compared to cells treated with antimycin A alone. Conclusion: The new antioxidant DHLHZn may have potential for therapeutic application in liver I/R injury, although this is a limited animal study.     

Endotoxin tolerance protects against local hepatic ischemia/reperfusion injury in the rat

Liver surgery and liver transplantation as well as circulatory shock are often associated with hepatic ischemia/reperfusion (I/R) injury. Recent evidence suggests that TNF-alpha plays a central role in I/R injury and, therefore, down-regulation of TNF-alpha seems to be a promising way to protect against the deleterious consequences of I/R. Endotoxin tolerance represents a state of unresponsiveness to endotoxin and is associated with diminished TNF-alpha production. Thus, the effect of endotoxin tolerance on hepatic I/R injury of the liver was investigated in a rat model. I/R injury was induced by temporary ischemia of the left lateral liver lobe for 90 min followed by a 3 h observation period of reperfusion. I/R injury resulted in functional hepatic disorder characterized by a decrease both in bile flow and bile acid concentration and 50% mortality. This was prevented by induction of endotoxin tolerance. Hepatic TNF-alpha mRNA expression after I/R of the liver was determined by RT-PCR. In untreated rats, TNF-alpha mRNA was induced in the liver 60 min after reperfusion and further increased until 3 h after reperfusion. In contrast, in endotoxin-tolerant rats, no increases in TNF-alpha mRNA expression were detected. This suggests that induction of endotoxin tolerance protects against hepatic I/R injury possibly via down-regulation of intra-organ TNF-alpha expression.     

Protective effect of curcumin against liver warm ischemia/reperfusion injury in rat model is associated with regulation of heat shock protein and antioxidant enzymes

AIM： To investigate the hypothesis that the protective effects of curcumin in hepatic warm ischemia/reperfusion （I/R） injury are associated with increasing heat shock protein 70 （Hsp70） expression and antioxidant enzyme activity. METHODS： Sixty Sprague-Dawley male rats were randomly divided into sham, I/R, C ＋ I/R groups. The model of reduced-size liver warm ischemia and reperfusion was used. Curcumin （50 mg/kg） was administered by injection through a branch of superior mesenteric vein at 30 min before ischemia in C ＋ I/R group. Five rats were used to investigate the survival during 1 wk after operation in each group. Blood samples and liver tissues were obtained in the remaining animals after 3, 12, and 24 h of reperfusion to assess serum alanine aminotransferase （ALT）, aspartate aminotransferase （AST）, liver tissue NO2- ＋ NO3-, malondialdehyde （MDA） content, superoxide dismutase （SOD）, catalase （CAT）, nitricoxide synthase （NOS） and myeloperoxidase （MPO） activity, HspT0 expression and apoptosis ratio. RESULTS： Compared with I/R group, curcumin pretreatment group showed less ischemia/reperfusioninduced injury. CAT and SOD activity and Hsp70 expression increased significantly. A higher rate of apoptosis was observed in I/R group than in C ＋ I/R group, and a significant increase of MDA, NO2^- ＋ NO3^- and MPO level in liver tissues and serum transaminase concentration was also observed in I/R group compared to C ＋ I/R group. Curcumin also decreased the activity of inducible NO synthase （iNOS） in liver after reperfusion,but had no effect on the level of endothelial NO synthase （eNOS） after reperfusion in liver. The 7 d survival rate was significantly higher in C ＋ I/R group than in I/R group. CONCLUSION： Curcumin has protective effects against hepatic I/R injury. Its mechanism might be related to the overexpression of Hsp70 and antioxidant enzymes.     

Resveratrol attenuates oxidative stress and histological alterations induced by liver ischemia/reperfusion in rats

AIM： To investigate the effects of resveratrol on liver ischemia/reperfusion （I/R） injury in rats. METHODS： A total of 40 male Sprague-Dawley rats weighing 240-290 g were randomized into four groups of ten： （1） controls： data from unmanipulated animals; （2） sham group： rats subjected to the surgical procedure, except for liver I/R, and given saline; （3） I/R group： rats underwent liver ischemia for 45 rain followed by reperfusion for 45 rain; （4） I-R/Resveratrol group： rats pretreated with resveratrol （10 umol/L, iv）. Liver tissues were obtained to determine antioxidant enzyme levels and for biochemical and histological evaluation. RESULTS： Plasma aminotransferase activities were higher in the I/R group than in the I-R/Resveratrol group. Malondialdehyde levels and the hepatic injury score decreased, while superoxide dismutase, catalase, and glutathione peroxidase levels increased in group 4 compared to group 3. In group 4, histopathological changes were significantly attenuated in resveratroltreated livers. CONCLUSION： These results suggest that resveratrol has protective effects against hepatic I/R injury, and is a potential therapeutic drug for ischemia reperfusionrelated liver injury.     

Glycine maintains mitochondrial activity and bile composition following warm liver ischemia-reperfusion injury

Background and Aim: Experimental studies have shown protective effect by the non‐essential amino acid glycine to liver ischemia‐reperfusion (I/R) injury but the mechanism of action is unknown. Methods: A rabbit model of hepatic lobar I/R was used. Three groups of animals (n = 6) were studied: Sham group (laparotomy alone), ischemia reperfusion (I/R) group (1 h of liver lobar ischemia and 6 h of reperfusion), and a glycine I/R group (intravenous glycine 5 mg/kg prior to the I/R protocol). Systemic and hepatic hemodynamics, degree of liver injury (bile flow, transaminases), hepatic microcirculation, mitochondrial activity (redox state of cytochrome oxidase), bile composition and cytokines (tumor necrosis factor‐α and interleukin‐8) were measured during the experiment. Results: Glycine administration increased portal blood flow, bile production, hepatic microcirculation and maintained cytochrome oxidase activity as compared with the I/R group during reperfusion. Glycine also reduced bile lactate surge and stimulated acetoacetate release in bile during reperfusion versus the I/R group. Cytokine levels (tumor necrosis factor‐α, interleukin‐8) and hepatocellular injury (aspartate aminotransferase and alanine aminotransferase) were significantly reduced by glycine administration. Conclusion: Intravenous glycine administration reduces liver warm I/R injury by reducing the systemic inflammatory response, and maintaining cellular energy production.     

NF-kB在大鼠肝缺血再灌注损伤中的活化及意义

目的探讨NFkB在肝缺血再灌注损伤过程中的作用。方法选择健康雌性Wistar大鼠24只,随机分为手术对照组,肝缺血90min组,肝缺血90min/再灌注120min组,每组8只。常规方法观察肝脏组织学改变,检测血清酶学水平和肝组织中髓过氧化物酶(MPO)含量,采用sABC免疫组织化学方法测定肝组织中NFkB的活化程度。结果手术对照组肝组织形态正常,无NFkB活化,肝功能酶学和MPO正常水平;缺血组动物肝细胞索排列紊乱,肝小叶变形,肝细胞和内皮细胞普遍水肿变性,NFkB呈中重度活化,血清酶学和MPO水平升高(P<0.01);肝缺血/再灌注组肝组织在缺血组改变基础上合并中央区局灶性肝细胞坏死,血窦内微血栓形成,汇管区中性粒细胞浸润,NFkB活化最为明显,血清酶学和MPO升高最为显著(P<0.01)。结论肝缺血再灌注时,NFkB被活化,使中性粒细胞组织浸润,对肝脏缺血再灌注损伤病理过程起到重要的作用。     

Splenectomy-reduced hepatic injury induced by ischemia/reperfusion in the rat

Abstract: In the present study, we investigated the role of the spleen in experimental hepatic ischemia/reperfusion in the rat. After a 90-min period of ischemia in the left and middle hepatic lobes, the ischemia was released and the liver was reperfused for up to 24 h. Plasma alanine aminotransferase reached a peak 3 h after the onset of reperfusion, and gradually decreased thereafter. A histological examination revealed evidence of hepatocellular necrosis and degeneration, especially 24 h after the onset of reperfusion. In addition, there was a noticeable accumulation of polymorphonuclear cells in the liver following ischemia/reperfusion. A splenectomy performed just prior to ischemia/reperfusion reduced both biochemical and histological hepatocellular injury. The number of polymorphonuclear cells in the liver following ischemia/reperfusion was significantly reduced in rats subjected to splenectomy, suggesting that the increase in polymorphonuclear cells may contribute to liver injury. The number of mononuclear cells also increased in the marginal zones of the spleen following ischemia/reperfusion, and appeared to be derived from the splenic monocyte/macrophage population, based on immunohistochemical studies. The spleen plays an important role in the pathogenesis of hepatic ischemia/reperfusion injury and the splenic monocyte/macrophage population contributes to liver damage.     

Cholestasis enhances liver ischemia/reperfusion‐induced coagulation activation in rats

Aim: Cholestasis is associated with increased morbidity and mortality in patients undergoing major liver surgery. An additional risk is induced when vascular inflow occlusion is applied giving rise to liver ischemia/reperfusion (I/R) injury. The role of the coagulation system in this type of injury is elusive. The aim of the current study was to assess activation of coagulation following hepatic I/R injury in cholestatic rats. Methods: Male Wistar rats were randomized into two groups and subjected to bile duct ligation (BDL) or sham laparotomy. After 7 days, both groups underwent 30 min partial liver ischemia. Animals were sacrificed before ischemia or after 6 h, 24 h, and 48 h reperfusion. Results: Plasma AST and ALT levels were higher after I/R in cholestatic rats (P < 0.05). Hepatic necrosis, liver wet/dry ratio and neutrophil influx were increased in the BDL group up to 48 h reperfusion (P < 0.05). Liver synthetic function was decreased in the BDL group as reflected by prolonged prothrombin time after 6 h and 24 h reperfusion (P < 0.05). I/R in cholestatic rats resulted in a 12‐fold vs. 7‐fold (P < 0.01) increase in markers for thrombin generation and a 6‐fold vs. 2‐fold (P < 0.01) increase in fibrin degradation products (BDL vs. control, respectively). In addition, the cholestatic rats exhibited significantly decreased levels of antithrombin (AT) III and increased levels of the fibrinolytic inhibitor plasminogen activator inhibitor (PAI‐1) during reperfusion. Conclusions: Cholestasis significantly enhances I/R‐induced hepatic damage and inflammation that concurs with an increased activation of coagulation and fibrinolysis.