活体生物荧光成像技术检测HO-1在HO-1/Luc小鼠肝脏缺血模型中的表达

目的尝试使用活体生物荧光成像技术无创定量检测HO-1在活体动物肝脏温缺血模型中的时空表达。方法建立小鼠部分肝脏温缺血模型，使用活体生物荧光成像技术连续检测HO-1在HO-1／Luc转基因报告小鼠中的转录情况。使用RT-PCR方法检测HO-1 mRNA水平，免疫组织化学方法行HO-1的表达定位。结果缺血肝叶发出的荧光信号最早于再灌注后3h被检测到，随后信号不断增强在9h达到峰值，然后信号逐渐减弱至再灌注后48h恢复至基础水平。RT-PCR方法测得在HO-1蛋白上调之前出现了HO-1mRNA的表达增强。免疫组织化学染色方法证实肝脏细胞是缺血再灌注损伤后HO-1表达上调的主要部位。结论活体生物荧光成像技术可以实时定量检测肝脏缺血后HO-1的表达，是一项敏感的检测技术。     

血红素氧合酶-1在HO-1/Luc小鼠原位肝脏移植模型中的表达

目的 应用活体生物萤光成像技术(BLT)无创定量检测血红素氧合酶-1(HO-1)在活体动物肝脏移植模型中的时空表达.方法 建立小鼠原位肝脏移植模型(HO-1/Luc转基因小鼠8只,wildtype小鼠40只),使用活体生物萤光成像技术连续检测HO-1在移植术后HO-1/Luc转基因小鼠中的转录.应用逆转录-聚合酶链反应(RT-PCR)检测HO-1 mRNA水平,免疫组织化学方法 (IHC)行HO-1的表达定位.结果 移植肝脏发出的萤光信号最早于移植后1 h被检测到(P<0.05),随后信号不断增强在6 h达到峰值.与移植术前比较,除0、48 h外信号差异均有统计学意义(P<0.05).移植后48 h信号衰减至基础水平.与移植术前比较,RT-PCR方法 测得HO-1 mRNA于0～9 h均显著升高(P<0.05),其中于3 h达到峰值,12 h降至基础水平.IHC证实肝脏细胞是移植后HO-1表达上调的主要部位.结论 活体生物萤光成像技术可实时定量检测肝脏移植后HO-1的表达.     

Toll-Like Receptor and Heme Oxygenase-1 Signaling in Hepatic Ischemia/Reperfusion Injury

Ischemia/reperfusion injury (IRI) represents the major problem in clinical liver transplantation. We have shown that toll-like receptor 4 (TLR4) signaling is specifically required in initiating antigen-independent IRI leading to liver inflammation, whereas local induction of anti-oxidant heme oxygenase-1 (HO-1) is cytoprotective. This study analyzes in vivo interactions between HO-1 and sentinel TLR system in the pathophysiology of liver IRI. Using a 90-min lobar warm ischemia model, wild type (WT), TLR4 KO/mutant and TLR2 KO mice were first assessed for the severity of hepatocellular damage at 6 h postreperfusion. Unlike in WT or TLR2-deficient mice, disruption/absence of TLR4 pathway reduced IRI, as manifested by liver function (serum alanine aminotransferase levels), histology (Suzuki's scores), neutrophil infiltration (myeloperoxidase activity) and local/systemic TNF-alpha production (mRNA/protein levels). Moreover, defective TLR4 but not TLR2 signaling increased mRNA/protein HO-1 expression. In contrast, tin protoporphyrin-mediated HO-1 inhibition restored hepatic damage in otherwise IRI-resistant TLR4 mutant/KO mice. CoPP-induced HO-1 overexpression ameliorated hepatic damage in IRI-susceptible TLR2 KO mice, comparable with WT controls, and concomitantly diminished TLR4 levels. In conclusion, this study highlights the importance of cross talk between HO-1 and TLR system in the mechanism of hepatic IRI. Hepatic IRI represents a case for innate immunity in which HO-1 modulates proinflammatory responses that are triggered via TLR4 signaling, a putative HO-1 repressor.     

Heme oxygenase-1 induction by the clinically used anesthetic isoflurane protects rat livers from ischemia/reperfusion injury

OBJECTIVE: It was the aim of this study to characterize the influence of isoflurane-induced heme oxygenase-1 (HO-1) expression on hepatocellular integrity after ischemia and reperfusion. SUMMARY BACKGROUND DATA: Abundant experimental data characterize HO-1 as one of the most powerful inducible enzymes that contribute to the protection of the liver and other organs after harmful stimuli. Therapeutic strategies aimed at utilizing the protective effects of HO-1 are hampered by the fact that most pharmacological inducers of this enzyme perturb organ function by themselves and are not available for use in patients because of their toxicity and undesirable or unknown side effects. METHODS: Rats were pretreated with isoflurane before induction of partial hepatic ischemia (1 hour) and reperfusion (1 hour). At the end of each experiment, blood and liver tissue were obtained for molecular biologic, histologic, and immunohistochemical analyses. RESULTS: Isoflurane pretreatment increased hepatic HO-1 mRNA, HO-1 protein, HO enzyme activity, and decreased plasma levels of AST, ALT, and alpha-GST. Histologic analysis of livers obtained from isoflurane-pretreated rats showed a reduction of necrotic areas, particularly in the perivenular region, the predominant site of isoflurane-induced HO-1 expression. In addition, sinusoidal congestion that could otherwise be observed after ischemia/reperfusion was inhibited by the anesthetic. Furthermore, isoflurane augmented hepatic microvascular blood flow and lowered the malondialdehyde content within the liver compared with control animals. Administration of tin protoporphyrin IX inhibited HO activity and abolished the isoflurane-induced protective effects. CONCLUSIONS: This study provides first evidence that pretreatment with the nontoxic and clinically approved anesthetic isoflurane induces hepatic HO-1 expression, and thereby protects rat livers from ischemia/reperfusion injury.     

Expression of Heme Oxygenase-1 in Human Livers Before Transplantation Correlates with Graft Injury and Function After Transplantation

Upregulation of heme oxygenase-1 (HO-1) has been proposed as an adaptive mechanism protecting against ischemia/reperfusion (I/R) injury. We investigated HO-1 expression in 38 human liver transplants and correlated this with I/R injury and graft function. Before transplantation, median HO-1 mRNA levels were 3.4-fold higher (range: 0.7-9.3) in donors than in normal controls. Based on the median value, livers were divided into two groups: low and high HO-1 expression. These groups had similar donor characteristics, donor serum transaminases, cold ischemia time, HSP-70 expression and the distribution of HO-1 promoter polymorphism. After reperfusion, HO-1 expression increased significantly further in the initial low HO-1 expression group, but not in the high HO-1 group. Postoperatively, serum transaminases were significantly lower and the bile salt secretion was higher in the initial low HO-1 group, compared to the high expression group. Immunofluorescence staining identified Kupffer cells as the main localization of HO-1. In conclusion, human livers with initial low HO-1 expression (<3.4 times controls) are able to induce HO-1 further during reperfusion and are associated with less injury and better function than initial high HO-1 expression (>3.4 times controls). These data suggest that an increase in HO-1 during transplantation is more protective than high HO-1 expression before transplantation.     

Change and role of heme oxygenase-1 in injured lungs following limb ischemia／reperfusion in rats

Lowerlimbischemiafollowedbyreperfusionisanimportantandcommonclinicalevent .Bothclinicalobservationandanimalexperimentindicatethatrestorationofbloodflowcansavethelimbsbutresultsinmultisystemorgandysfunctionevendeath .1Althoughthesystemicinflammationoflimbischemia/reperfusion (I/R )candamageanyorgan ,theonsetofthesyndromeisusuallyheraldedbythedevelopmentof pulmonarydysfunction .2 ,3Thiskindofpulmonarydysfunctionischaracterizedbyincreasedlungvascular permeabilityandpulmonaryhypertension ,whichis…     

Gadolinium chloride-induced improvement of postischemic hepatic perfusion after warm ischemia is associated with reduced hepatic endothelin secretion

Selective Kupffer cell blockade by gadolinium chloride (GdCl₃) pretreatment of liver donors previously proved to be effective in reducing ischemia/reperfusion injury in rat liver transplants. Physiological mechanisms of this effect have not been specified so far. Vasoactive peptides are involved in liver blood flow regulation. We tested the hypothesis, that hepatic hemodynamic effects of GdCl₃ pretreatment are mediated by intrahepatic endothelin‐1 (ET) secretion in a standardized porcine model of warm liver ischemia and reperfusion. Standardized warm hepatic ischemia (45 min) was induced after laparotomy in intubation narcoses (ITN) by Pringle‐maneuver in pigs (n = 12). Animals were either pretreated with GdCl₃ (20 mg/kg i.v.) or sodium chloride 0.9% (control group) in a randomized manner 24 h before investigation. Relaparotomy was performed at day 7. Before, during ischemia and until 6 h after liver reperfusion, transhepatic blood flow (portal venous + hepatic artery flow) was defined by ultrasonic flow probes and hepatic parenchymous microcirculation evaluated by implanted thermodiffusion electrodes. ET plasma concentrations were analyzed (commercial RIA) at all time points in the hepatic veins after selective canulation. GdCl₃ pretreatment of animals markedly improved hepatic macro‐ and microperfusion before and particularly after warm ischemia. Mean ET plasma concentrations in the hepatic vein were significantly lower before, 6 h and 7 days after ischemia, compared with controls. Kupffer cell destruction by GdCl₃ pretreatment improves hepatic micro‐ and macroperfusion after warm ischemia, thus indicating reduced ischemia/reperfusion injury. Documented reduction of postischemic liver blood flow impairment after GdCl₃ pretreatment could be mediated by a decreased hepatic ET secretion, as hemodynamic effects were associated with significantly reduced ET plasma levels in hepatic veins.     

Alleviating ischemia-reperfusion injury in aged rat liver by induction of heme oxygenase-1

BACKGROUND: Heme oxygenase-1 (HO-1), a cytoprotective protein, may be important in ameliorating hepatic ischemia-reperfusion (I/R) injury, a critical factor in the dysfunction of the aged liver after transplantation. METHODS: We used hemin to overexpress HO-1 and analyze its effects in a model of I/R in aged livers used for orthotopic transplantation. RESULTS: The SGOT levels in the hemin group were significantly lower than those of the saline treatment group. Hemin liver grafts showed markedly fewer apoptotic (TUNEL+) liver cells after reperfusion compared with the controls. The plasma nitric oxide levels in the hemin group were significantly lower than those in the control group. Unlike untreated or hemin + Znpp-treated orthotopic liver transplant controls, iNOS expression in the hemin group was almost absent at 12 and 24 hours, after reperfusion. In contrast, eNOS was comparable in hemin and saline orthotopic liver transplants. The increased levels of Bcl-2 expression compared with saline controls were most pronounced at 12 hours after transplantation. In contrast, caspase 3 was lower at 24 hours among the hemin-pretreated group compared with saline-treated liver transplant controls. CONCLUSIONS: HO-1 alleviated the I/R injury in the aged liver by suppressing local expression of inducible nitric oxide synthase and by modulating pro- and antiapoptotic pathways.     

Role of EP4 prostaglandin E2 receptor in the ischemic liver

Prostaglandin E(2) (PGE(2)) mediates a variety of both innate and adaptive immunity responses through 4 distinct receptors, EP1-4. Recent studies have suggested the physiological and pathological role of EP4 in various inflammatory diseases. In this study, we investigated the importance of the EP4 receptor, and the efficacy of a selective EP4 agonist to alter hepatic ischemia/reperfusion (I/R) injury, an important cause of damage in liver resection and transplantation. We used an established murine I/R injury model, 70% partial hepatic ischemia for 90 minutes in male C57BL/6 mice. The local expression of EP4 messenger RNA (mRNA) in the naive and the ischemic liver at 2 hours after reperfusion was examined using RT-PCR analysis. Some mice received the EP4 selective agonist during I/R. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured as markers of hepatic injury. EP4 expression in the liver was significantly up-regulated at 2 hours after reperfusion. Furthermore, treatment with EP4 agonist significantly inhibited hepatic injury at 6 hours after reperfusion. Our data suggest an inhibitory role of EP4 PGE(2) receptor in hepatic I/R injury and the therapeutic efficacy of a selective EP4 agonist for liver protection.     

血红素氧合酶-1在临床肝移植中肝脏保护作用的研究

目的 研究移植肝脏血红素氧合酶(HO-1)表达水平与缺血再灌注损伤和移植术后肝脏功能的关系.方法 研究28例人类临床原位肝脏移植,根据供肝血红素氧合酶(HO-1)表达的平均值将供肝分为两组:移植前供肝HO-1高表达组和移植前供肝HO-1低表达组.比较两组移植术后血浆AST、ALT水平、胆汁中胆盐含量以及术前术后HO-1 mRNA和蛋白表达情况.结果 再灌注后移植术前HO-1低表达组的HO-1 mRNA表达显著增加,而高表达组HO-1 mRNA表达却有所下降.肝脏移植后,术前HO-1低表达组与高表达组相比,血浆转氨酶显著降低,胆汁中胆盐含量明显高于后者.结论 移植术前HO-1低表达组供肝在再灌注过程中能够进一步诱导HO-1表达,与高表达组供肝相比其所遭受的缺血再灌注损伤较轻,移植术后肝脏功能较好.移植过程中HO-1表达的增强要比移植前HO-1高表达更具有细胞保护作用.免疫荧光染色证实枯否细胞是人类肝脏表达HO-1的主要部位.     

上调肝脏HO-1抑制肥大细胞脱颗粒减轻大鼠肝脏缺血再灌注损伤

目的 探索HO-1对肝脏缺血再灌注损伤中肥大细胞脱颗粒的影响。方法 将20只SD大鼠随机分成4组：假手术组（Sham组）,缺血再灌注损伤组（I/RI组）,HO-1诱导剂钴原卟啉组(CoPP组,术前24h给予CoPP,5 mg/kg)及HO-1抑制剂锌原卟啉组(ZnPP组,术前24h给予ZnPP,20 mg/kg)。建立大鼠缺血再灌注损伤模型,各组于再灌注后2h收集标本。RT-PCR检测肝脏组织HO-1 mRNA表达,Western blot检测肝脏组织HO-1蛋白表达;测定血清中ALT、AST水平;肝脏组织甲苯胺蓝染色检测肥大细胞脱颗粒数量,HE染色评价肝脏组织损伤情况。结果 与Sham组相比,I/RI组、CoPP组、ZnPP组大鼠组织HO-1 RNA和蛋白表达增加,血清ALT、AST水平升高,肥大细胞脱颗粒数量增多,肝脏细胞损伤加重。CoPP组与I/RI组相比,HO-1 mRNA和蛋白表达增加,血清ALT、AST水平减低,肥大细胞脱颗粒数量减少,肝细胞损伤减轻。ZnPP组与I/RI组相比,HO-1 mRNA和蛋白表达减少,血清ALT、AST水平升高,肥大细胞脱颗粒数量增多、肝细胞损伤严重。组间比较差异具有统计学意义(P＜0.05)。结论 HO-1过表达能减轻肝脏I/RI,其机制可能与抑制肝脏组织中肥大细胞脱颗粒有关。     

Reduction of hepatic ischemia/reperfusion injury by a soluble P-selectin glycoprotein ligand-1.

OBJECTIVE: The authors' goal was to determine the effects of specific binding and blockade of P- and E-selectins by a soluble P-selectin glycoprotein ligand-1 (PSGL-1) in rat models of hepatic in vivo warm ischemia and ex vivo cold ischemia. The authors also sought to determine the effect of selectin blockade on isograft survival in a syngeneic rat orthotopic liver transplant model. SUMMARY BACKGROUND DATA: Ischemia/reperfusion (I/R) injury is a major factor in poor graft function after liver transplantation, which may profoundly influence early graft function and late changes. It is hypothesized that I/R injury leads to the upregulation of P-selectin, which is then rapidly translocated to endothelial cell surfaces within 5 minutes of reperfusion of the liver, initiating steps leading to tethering of polymorphonuclear neutrophil leukocytes to the vascular intima. Local production by leukocytes of interleukin-1, tumor necrosis factor-alpha, or both induces P-selectin expression on the endothelium and continues the cascade of events, which increases cell adherence and infiltration of the organ. METHODS: To examine directly the effects of selectins in a warm hepatic I/R injury model, 100 microg of PSGL-1 or saline was given through the portal vein at the time of total hepatic inflow occlusion. The effects of PSGL-1 in cold ischemia were assessed using an isolated perfused rat liver after 6 hours of 4 degrees C storage in University of Wisconsin (UW) solution, with or without the instillation of PSGL-1 before the storage. To evaluate the effect of selectin blockade on liver transplant survival, syngeneic orthotopic liver transplants were performed between inbred male Sprague-Dawley rats after 24 hours of cold ischemic storage in UW solution. A separate group of animals received two doses of 100 microg of PSGL-1 through the portal vein before storage and before reperfusion of the transplanted liver. Recipient survival was assessed at 7 days, and the Kaplan-Meier product limit estimate method was used for univariate calculations of time-dependent recipient survival events. RESULTS: In an in vivo warm rat liver ischemia model, perfusion with PSGL-1 afforded considerable protection from I/R injury, as demonstrated by decreased transaminase release, reduced histologic hepatocyte damage, and suppressed neutrophil infiltration, versus controls (p < 0.05). When cold stored livers were reperfused, PSGL-1 reduced the degree of hepatocyte transaminase release, reduced neutrophil infiltration, and decreased histologic hepatocyte damage (p < 0.05 vs. UW-only controls). On reperfusion, livers treated with PSGL-1 demonstrated increased portal vein blood flow and bile production (p < 0.05 vs. UW-only controls). In addition, 90% of the rats receiving liver isografts stored in UW solution supplemented with PSGL-1 survived 7 days versus 50% of those whose transplanted syngeneic livers had been stored in UW alone (p < 0.05). CONCLUSIONS: Selectins play an important role in I/R injury of the liver. Early modulation of the interaction between P-selectin and its ligand decreases hepatocyte injury, neutrophil adhesion, and subsequent migration in both warm and cold rat liver ischemia models. In addition, the use of PSGL-1 before ischemic storage and before transplantation prevents hepatic injury, as documented by a significant increase in liver isograft survival. These findings have important clinical ramifications: early inhibition of alloantigen-independent mechanisms during the I/R damage may influence both short- and long-term survival of liver allografts.     

Pyrrolidine dithiocarbamate provides protection against hypothermic preservation and transplantation injury in the rat liver: the role of heme oxygenase-1

BACKGROUND: Pyrrolidine dithiocarbamate (PDTC) represents a class of antioxidants and is a potent inducer of the heme oxygenase-1 (HO-1) gene and an inhibitor of nuclear factor-kappa B (NF-kappa B). We examined the impact of PDTC preconditioning against cold ischemia and reperfusion injury in the rat liver. METHODS: Lewis rats were treated subcutaneously with saline or PDTC solution 24 hours before harvesting. Some animals pretreated with PDTC were also given zinc protoporphyrin IX intravenously immediately after reperfusion. HO-1 expression and enzyme activity in liver tissues were analyzed at different time points after each treatment. After transplantation of 24-hour preserved livers, serum levels of transaminases and gene expression of tumor necrosis factor-alpha, interleukin-1 beta, and NF-kappa B were measured. Animal survival and cellular viability were monitored. RESULTS: HO-1 gene expression and protein synthesis were enhanced in PDTC-treated livers, leading to increased enzyme activity (P <.05). The PDTC treatment group showed lower transaminase levels (P <.05), lower cytokine and NF-kappa B messenger RNA expression (P <.05), and fewer nonviable cells (P <.05) than did the control group, whereas these PDTC effects were abolished with zinc protoporphyrin injection after reperfusion (P <.05). The best animal survival rate was observed in the PDTC group (P <.05). CONCLUSION: PDTC preconditioning reduces inflammatory responses during reperfusion. PDTC appears to exert this protective effect by induction of an antioxidative stress protein and inhibition of proinflammatory cytokines.     

Initial hepatic microcirculation correlates with early graft function in human orthotopic liver transplantation.

Microcirculatory disturbances are an initial causative determinant in hepatic ischemia/reperfusion injury. The aim of this study was to assess sinusoidal perfusion during human liver transplantation using orthogonal polarization spectral imaging and to evaluate the significance of intraoperative microcirculation for early postoperative graft function. Hepatic microcirculation was measured in 27 recipients undergoing full-size liver transplantation and compared to a group of 32 healthy living-related liver donors. The microvascular parameters were correlated with postoperative aspartate aminotransferase and bilirubin levels. Hepatic perfusion following liver transplantation was found to be significantly decreased when compared with the control group. Volumetric blood flow within the individual sinusoids increased due to sinusoidal dilatation and enhanced flow velocity. Regression analysis of postoperative aspartate aminotransferase and bilirubin with microvascular parameters revealed significant correlations. The extent of volumetric blood flow increased within the first 30 minutes after reperfusion and showed a significant correlation with postoperative aspartate aminotransferase release and bilirubin elimination. In conclusion, postischemic hepatic microvascular perfusion was analyzed in vivo, demonstrating significant microvascular impairment during liver transplantation. Sinusoidal hyperperfusion appears to confer protection against postischemic liver injury, as given by the correlation with aspartate aminotransferase and bilirubin levels. Thus, these findings may have therapeutic importance with respect to mechanisms mediating postischemic reactive hyperemia.     

HO-1 upregulation suppresses type 1 IFN pathway in hepatic ischemia/reperfusion injury

Upregulation of heme oxygenase (HO)-1, a heat shock protein 32, protects against hepatic ischemia/reperfusion (I/R) injury. Activation of "innate" toll-like receptor (TLR) 4 system triggers the I/R injury cascade. This study explores cytoprotective functions of HO-1 overexpression following exogenous administration of cobalt protoporphyrin (CoPP), and its relationship with the TLR4 pathway in a model of mouse partial hepatic warm I/R injury. CoPP treatment markedly improved hepatic function and histology, and suppressed pro-inflammatory cytokine elaboration profile, as compared with untreated controls. Although administration of CoPP did not affect intrahepatic TLR4, it downregulated IFN-inducible protein 10 (IP-10) expression. As IP-10 is the major product of type-1 IFN pathway downstream of TLR4, we then infused recombinant IFN-beta (rIFN-beta) directly into mouse livers. Interestingly, infusion of rIFN-beta upregulated hepatic IP-10 expression. In contrast, adjunctive CoPP treatment decreased IP-10 levels in mouse livers infused with rIFN-beta. Thus, CoPP-induced HO-1 upregulation suppresses type-1 IFN pathway downstream of TLR4 system in hepatic warm I/R injury model.     

血红素加氧酶-1诱导对鼠肝缺血再灌注损伤的保护作用

目的研究血红素加氧酶-1(heme oxygenase-1,HO-1)在鼠肝缺血再灌注损伤肝组织中的表达及其作用。方法建立小鼠部分肝脏热缺血再灌注损伤模型,36只清洁级Balb/C小鼠随机分为3组: 假手术组(S组)、缺血/再灌注损伤组(I/R组)、HO-1诱导剂氯化高铁血红素(hemin)预处理组(HM组)。免疫组化半定量分析肝组织HO-1蛋白的表达,检测血清AST和ALT,肝组织丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性,并观察肝组织的病理变化。结果与S组比较,I/R组HO-1蛋白表达显著增强,hemin预处理后,HO-1蛋白表达较I/R组增高(P<0．01)。I/R组AST,ALT活性和MDA的含量显著高于S组,而HM组均显著低于I/R组(P<0．01);I/R组SOD活性下降,而HM组显著高于I/R组(P<0．01)。HM组病理损伤程度明显轻于I/R组。结论 HO-1在鼠肝缺血再灌注损伤肝组织中表达上调,对肝脏具有保护效应。     

Thrombospondin-1 mRNA expression in experimental kidney transplantation with heart-beating and non-heart-beating donors

BACKGROUND: Ischemia-reperfusion (IR) is a risk factor for delayed graft function, a clinical syndrome more frequently observed in non-heart-beating donors (NHBDs). Previous studies have reported that transforming growth factor-beta1 (TGF-beta1) and hypoxia-inducible factor-1alpha (HIF-1alpha) gene expression increase in the first few days after kidney transplant and that this increase in TGF-beta1 expression is lower in NHBD animals. The purpose of this study was to extend the gene profile analysis by characterizing TGF-beta1 activator thrombospondin-1 (TSP-1) and genes related to HIF-1alpha such as heme oxygenase-1 (HO-1), nitric oxide synthase-2 (NOS-2) and NOS-3. METHODS: The experimental pig model of kidney transplantation comprised heart-beating donors (HBDs, n=9) and NHBDs (n=22). Cortical biopsies were collected after anesthetic induction (baseline), after warm ischemia (WI), after cold ischemia (CI), after 1 hour of reperfusion (1R) and 5 days (5D) after transplant. TSP-1, HO-1, NOS-2 and NOS-3 mRNA expression was determined by real-time PCR. RESULTS: No change in expression of any of the genes analyzed was found during the transplant procedure (WI, CI, 1R) in HBD and NHBD cortical samples. TSP-1 mRNA was significantly increased at 5D in NHBD animals but unchanged in the HBD group. HO-1 was up-regulated in HBD (p<0.05) and NOS-2 mRNA was significantly increased in both groups (p<0.05). No difference in NOS-3 expression was observed at 5D. CONCLUSIONS: The increased TSP-1 expression in NHBDs may indicate a compensatory response to the reported diminished TGF-beta1 expression. The augmented NOS-2 and HO-1 expression in HBDs could have a positive effect on the recovery of kidney function.     

Prevention of ischemia-reperfusion-induced hepatic microcirculatory disruption by inhibiting stellate cell contraction using rock inhibitor

BACKGROUND: We demonstrated that hepatic stellate cells (HSCs) isolated from rat livers exposed to warm ischemia are significantly contractile when compared with HSCs from intact rat livers. This suggests that ischemia-reperfusion (IR)-induced impairment of sinusoidal microcirculation results, at least in part, from contraction of HSCs. METHODS: Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) is one of the key regulators of HSCs motility. Therefore we investigated whether Y-27632, a p160ROCK-specific inhibitor, has beneficial effects on warm IR injury in an in vivo rat partial liver IR model and a rat orthotopic liver transplantation model. RESULTS: After reperfusion following 90 min of warm ischemia, livers in untreated control rats had persistent congestion and impaired mitochondrial respiration, as demonstrated by increasing deoxy-hemoglobin and reduced cytochrome oxidase contents in the hepatic tissues using in vivo near-infrared spectroscopy. Serum levels of transaminase and endothelin (ET)-1 in these rats were markedly increased 1 hr after reperfusion. In contrast, when Y-27632 (3-30 mg/kg) was administered orally, hepatic tissue contents of deoxy-hemoglobin and cytochrome oxidase rapidly normalized. In such animals, the elevation of serum transaminase levels, but not that of ET-1 levels, was significantly suppressed. This is consistent with in vitro data demonstrating that Y-27632 causes HSCs to undergo relaxation even in the presence of ET-1. Moreover, in a rat orthotopic liver transplantation model, Y-27632 pretreatment dramatically improved the survival of recipients with liver grafts subjected to 45 min of warm ischemia. CONCLUSIONS: Y-27632 attenuates IR-induced hepatic microcirculation disruption by inhibiting contraction of HSCs.     

Beneficial effect of allopurinol in liver ischemia

The effect of allopurinol on protein synthesis, tissue water, and adenine nucleotides in liver tissue during and after a period of liver ischemia was investigated in rats. Ischemia was induced in the left and median liver lobes for 1 hour and experiments were continued for 2 hours after reperfusion. One group of animals (n = 20) received allopurinol (50 mg/kg body weight) intravenously 10 minutes before induction of liver ischemia. Control rats (n = 20) were given a corresponding volume of saline solution. Protein synthesis was measured by determining the rate of amino acid incorporation into protein in incubated liver slices. The reduction of protein synthesis and energy level in liver tissue and the increase of hepatic tissue water were similar in both groups of animals at the end of the ischemic period. During reperfusion the protein synthesis rate was higher and hepatic tissue water was lower in allopurinol-treated animals than in control rats. No significant differences in hepatic adenine nucleotides were found between the two groups of rats during ischemia or after reperfusion. The results demonstrated that improved protein synthesis and reduced tissue water in the postischemic liver after administration of allopurinol were not the result of improved restoration of adenine nucleotides. Inhibited production of oxygen-free radicals might be one mechanism by which allopurinol exerted its beneficial effect after liver ischemia.