The tyrosine kinase inhibitor tyrphostin AG126 reduces renal ischemia/reperfusion injury in the rat

BACKGROUND: We investigate the effects of tyrphostin AG126, an inhibitor of tyrosine kinase activity, on the renal dysfunction and injury caused by ischemia/reperfusion (I/R) of the kidney. METHODS: Tyrphostin AG126 (5 mg/kg intraperitoneally) was administered to male Wistar rats 30 minutes prior to bilateral renal ischemia for 45 minutes followed by reperfusion for up to 48 hours. Biochemical markers of renal dysfunction and injury were measured and renal sections assessed for renal injury. Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and formation of nitrotyrosine and poly (ADP) ribose (PAR) were assessed using immunohistochemistry. Rat proximal tubular cells (PTCs) were incubated with interferon-gamma (100 IU/mL), bacterial lipopolysaccharide (10 microg/mL), and with increasing concentrations of tyrphostin AG126 (0.0001-1 mmol/L) for 24 hours. Nitric oxide production was measured in both plasma from rats subjected to I/R and in incubation medium from PTCs. RESULTS: After 6 hours of reperfusion, tyrphostin AG126 significantly reduced the increase in serum and urinary indicators of renal dysfunction and injury caused by I/R and reduced histologic evidence of renal injury. Tyrphostin AG126 also improved renal function (after 24 and 48 hours of reperfusion) and reduced the histologic signs of renal injury (after 48 hours of reperfusion). Tyrphostin AG126 reduced the expression of iNOS and nitric oxide levels in both rat plasma and in PTC cultures, as well as expression of COX-2. Tyrphostin AG126 also reduced nitrotyrosine and PAR formation, suggesting reduction of nitrosative stress and poly (ADP-ribose) polymerase (PARP) activation, respectively. CONCLUSION: Taken together, these results show that tyrphostin AG126 significantly reduces the renal dysfunction and injury caused by I/R of the kidney. We propose that inhibition of tyrosine kinase activity may be useful against renal I/R injury.     

NFκB抑制剂对大鼠肝脏移植再灌注损伤的保护作用

目的　探讨大鼠肝脏移植后核因子κB(NFκB)活化对炎性介质表达和中性粒细胞浸润集聚的影响。方法　供受体鼠分别于术前 15min腹腔注射NFκB抑制剂脯氨酸二硫代氨基甲酸酯 (ProDTC 15mg/kg)。 结果　与对照组相比 ,应用ProDTC者移植后NFκBp6 5含量、肿瘤坏死因子 α(TNF α)、巨噬细胞炎性蛋白 2 (MIP 2 )、细胞间粘附分子 1(ICAM 1)mRNA表达和蛋白表达明显下降 ;髓过氧化物酶 (MPO)活性明显减低 (P均 <0 0 1)。ProDTC也显著降低天门冬氨酸转氨酶(AST)、丙氨酸转氨酶 (ALT)、乳酸脱氢酶 (LDH)水平 (P <0 0 1)。结论　ProDTC抑制移植后NFκB活化从而下调了TNF α、MIP 2、ICAM 1表达从而减轻中性粒细胞浸润及肝脏缺血再灌注损伤。     

P-selectin and chemokine response after liver ischemia and reperfusion

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

Urinary trypsin inhibitor ameliorates renal tissue oxygenation after ischemic reperfusion in rats

PURPOSE: In order to determine the mechanism of the protective effect of a urinary trypsin inhibitor (UTI) on renal ischemic reperfusion injury, we measured the tissue oxygen partial pressure pO2 in both the renal cortex and medulla in rats, using electron paramagnetic resonance (EPR) oximetry. METHODS: We allocated the rats to three groups: normal saline (NS) group, a UTI 50,000 U x kg(-1) (LD) group, and a UTI 150,000 U x kg(-1) (HD) group, with the normal saline and UTI being administered 30 min before ischemia. Renal ischemia was achieved by inflating the balloon of a vascular occluder that had been placed around the abdominal aorta just above the bifurcation of the renal artery. Cortical and medullary pO2 were measured every 10 min during ischemia (30 min) and reperfusion (60 min) by EPR oximetry; also, systemic cardiopulmonary parameters were measured. RESULTS: The pO2 in the cortex and medulla decreased to less than 2 mmHg during ischemia in all groups. At 60 min after reperfusion, the pO2 values in the NS group were not fully restored, whereas those in the LD and HD groups were completely restored to the pre-ischemic values. There were no significant differences between the HD and LD groups. There were no differences between any groups in cardiopulmonary parameters. CONCLUSION: Because UTI improved renal oxygenation after reperfusion without changing cardiopulmonary parameters, the pharmacological properties of UTI, such as its renal protection and anti-shock activity, may be explained in part, by this improvement in tissue oxygenation.     

L-Selectin and chemokine response after liver ischemia and reperfusion

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

Effect of specific neutrophil elastase inhibitor on ischemia/reperfusion injury in rat liver transplantation.

Activated neutrophils have been implicated as playing an important role in ischemia/reperfusion injury of the liver by releasing toxic mediators such as oxygen free radicals and elastases. In the present study, we evaluated the effect of a novel, specific neutrophil elastase inhibitor (ONO-5046) on cold-ischemia/reperfusion injury of the liver allograft in rodents. Livers from male Lewis rats were procured and stored cold (4 degrees C) in lactated Ringer's solution and transplanted orthotopically. Recipients were divided into three groups: Vehicle group, 5-h preservation and vehicle (n = 8); ONO-5046 group, 5-h preservation and administration of ONO-5046 (n = 8); and Control group, minimum preservation only (n = 8). Bile output after reperfusion was significantly larger in the ONO-5046 group compared to the Vehicle group (P < 0.05 or less). Sinusoidal endothelial cell function represented by the serum hyaluronic acid concentration at 120 min after reperfusion of the ONO-5046 group was significantly lower than that in the Vehicle group (17.0 +/- 7.9 vs 36.2 +/- 14.9 ng/ml, P < 0.05), whereas serum transaminase levels 120 min after reperfusion were comparable between the two groups. Liver tissue energy charge 120 min after reperfusion was significantly better in the ONO-5046 group compared to the Vehicle group (P < 0.05). Furthermore, the number of neutrophils infiltrating the allograft after reperfusion was significantly depressed in the ONO-5046 group compared to the Vehicle group (P < 0. 02). These data suggest that the neutrophil elastase might cause liver damage early after reperfusion in cold-stored liver, which can be ameliorated by the administration of a specific neutrophil elastase inhibitor, ONO-5046.     

Calpain inhibitor-1 reduces renal ischemia/reperfusion injury in the rat

BACKGROUND: Activation of the cysteine protease calpain has been implicated in renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of calpain inhibitor-1 (Cal I-1) in an in vivo model of renal I/R injury. METHODS: Male Wistar rats were administered Cal I-1 (10 mg/kg, IP) 30 minutes before undergoing bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Plasma concentrations of urea, creatinine, Na(+), gamma-glutamyl transferase (gamma GT), aspartate aminotransferase (AST) and urinary Na(+), glutathione S-transferase (GST), and N-acetyl-beta-D-glucosaminidase (NAG) were measured for the assessment of renal dysfunction and I/R injury. Creatinine clearance (C(Cr)) and fractional excretion of Na(+) (FE(Na)) were used as indicators of glomerular and tubular function, respectively. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of neutrophil infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). RESULTS: Cal I-1 significantly reduced I/R-mediated increases in urea, creatinine, gamma GT, AST, NAG, and FE(Na) and significantly improved C(Cr). Cal I-1 also significantly reduced kidney MPO activity and MDA levels. Cal I-1 also reduced histologic evidence of I/R-mediated renal damage and caused a substantial reduction in the expression of iNOS and COX-2, both of which involve activation of nuclear factor-kappa B (NF-kappa B). CONCLUSIONS:: These results suggest that Cal I-1 reduces the renal dysfunction and injury associated with I/R of the kidney. We suggest that the mechanism could involve the inhibition of I/R-mediated activation of NF-kappa B.     

Akt activation protects rat liver from ischemia/reperfusion injury

BACKGROUND: Apoptosis as well as necrosis may play an important role in hepatic ischemia/reperfusion (I/R) injury. Akt, a serine-threonine protein kinase, is known to promote cell survival. We investigated whether gene transfer of constitutively active or dominant negative Akt could affect hepatic I/R injury. MATERIALS AND METHODS: Hepatic I/R injury was induced in rats by Pringle's maneuver for 20 min followed by reperfusion. Adenoviruses encoding a constitutively active form of Akt (myrAkt), a dominant negative form of Akt (dnAkt), or beta-galactosidase (LacZ) were injected through the tail vein 72 h before hepatic I/R. RESULTS: Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining demonstrated a significant increase in the positive cells 240 min after reperfusion. Immunoblotting with phospho-Akt antibody showed phosphorylation of Akt from 90 to 180 min after reperfusion. The expression of myrAkt reduced the number of TUNEL-positive cells and hepatic necrosis around the central veins in the liver after reperfusion. This expression also significantly inhibited the increase in serum alanine aminotransferase (297 +/- 131 IU/L, P < 0.05) 120 min after I/R, compared with increases in uninfected (1761 +/- 671 IU/L), LacZ adenovirus (1528 +/- 671 IU/L)-, and dnAkt adenovirus (1342 +/- 485 IU/L)-infected rats. MyrAkt expression phosphorylated Bad and inhibited the release of cytochrome-c after reperfusion. No difference in nuclear translocation of nuclear factor (NF)-kappaB, p65 was seen among the three groups of rats, however. CONCLUSION: Adenoviral gene transfer of myrAkt could inhibit apoptotic cell death and subsequent hepatic I/R injury in the rat, through Bad, not NF-kappaB.     

ROCK inhibitor Y-27632 prevents primary graft non-function caused by warm ischemia/reperfusion in rat liver transplantation

Hepatic stellate cells (HSCs) can easily be activated by ischemia/reperfusion, and this activation results in hepatic microcirculatory disturbance by cell contraction. ROCK is one of the key regulators of the motility of HSCs, and Y-27632 suppresses the activation of HSCs. We examined whether Y-27632 treatment prevents primary graft non-function caused by 45-min warm ischemia in orthotopic liver transplantation (OLT). Donor and recipient rats were administered Y-27632 (3-30 mg/kg). Y-27632 treatment at 30 mg/kg in both donor and recipient prevented congestion of the grafted livers, as demonstrated by analysis of hemoglobin (Hb) content in the grafted livers, using in-vivo near-infrared spectroscopy. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hyaluronic acid at 4 h after OLT in the 30-mg/kg Y-27632-treated group were significantly lower than those in the control group. Specimens from the untreated control recipients showed sinusoidal congestion and massive fresh hepatocyte necrosis, whereas specimens from the Y-27632-treated recipients demonstrated minimal histological changes. Moreover, Y-27632 pre-treatment dramatically improved the survival of recipients. These results suggest that Y-27632 would be clinically useful for preventing liver failure associated with ischemia/reperfusion in liver transplantation.     

Neutrophil depletion and chemokine response after liver ischemia and reperfusion.

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

肝缺血再灌注对肝硬化大鼠的损伤作用

目的 研究肝硬化大鼠肝缺血再灌注（hepatic ischemia reperfusion,HIR）损伤的机制和程度。方法 用60％四经碳（CCl4）溶液皮下注射方法制作肝硬化大鼠模型，肝硬化大鼠随机分为六组：A组：假手术组（6只）；B、C、D组：分别为肝门完全阻断20min、30min、40min（每组各16只）；E组“单纯肠系膜上静脉阻断（16只）;F组：肝门阻断＋门腔转流（16只）；另外，随机取10只正常肝脏大鼠组成G组，行肝门完全阻断30min。观察7天存活率、丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、透明质酸（HA）、肿瘤坏死因子（TNF），以及肝、肺病理的变化。结果 B、C、D、E、F、G组的7天存活率分别为10／10只、6／10只、4／10只、5／10只、8／10只、6／10只；再灌注后4h血清TNF变化：后六组均明显高于术前，C、D组高于B、A组，E、F组也明显高于A组（P＜0.01）再灌注4h后HA变化：D组明显高于B、E组，F、C组明显高于A组（P＜0.05）；再灌注4h后D、C组的AST、ALT均明显高于B组、A组、D组的AST明显高于F组，F组的AST、ALT显著高于E组（P＜0.05）；C、G两组比较，上述指标的差异无显著性（P＞0.05）；肝、肺组织学检查可见肝、肺的病理损害，程度随缺血时间的延长而加重，E组损伤重于F组。结论 硬化肝脏肝缺血再灌注损伤涉及全身多个器官，门脉静淤血可能是损伤乃至死亡的主要原因；肝硬化大鼠耐受肝缺血的最大的时限在30min以内。     

核因子-κB/I-κB传导通路在肝脏缺血再灌注损伤中的作用

目的 探讨核因子(NF)—κB／Ⅰ—κB传导通路在肝脏缺血再灌注损伤中的作用。方法 采用阻断大鼠部分肝血供的缺血再灌注损伤模型，左半肝缺血90min，再灌注分0、1、2、4h等时点。用凝胶滞留电泳方法测定NF—κB的结合活性；应用逆转录—聚合酶链反应(RT—PCR)测定肝组织中肿瘤坏死因子—α(TNF—α)、细胞间粘附因子—1(ICAM—1)mRNA的表达量。结果 肝脏缺血再灌注损伤时NF—κB与其特异性调控序列的结合活性增高且具有时相性。再灌注1～2h NF—κB结合活性增高，4h后开始降低。肝组织中TNF—α、ICAM—1 mRNA表达在再灌注2h后升高。讨论 肝脏缺血再灌注损伤时，NF—κB激活并进入细胞核内，与一些炎症因子基因启动子区特异序列结合，上调TNF—α、ICAM—1 mRNA表达，从而引起肝脏缺血再灌注损伤。     

In vivo microscopy reveals that complement inhibition by C1‐esterase inhibitor reduces ischemia/reperfusion injury in the liver

Abstract Complement plays a decisive role in postischemic tissue injury, a process responsible for severe damage after organ ischemia. Several pathophysiologic mechanisms initiated upon reperfusion are mediated by complement inducing micro‐circulatory disturbances. Here, we demonstrate the effects of complement inhibition using C1‐esterase inhibitor (C1‐INH) on microcirculation after liver ischemia by invivo microscopy (IVM). In rats, the left liver lobe was clamped for 70 min. C1‐INH was given 1 min prior to reperfusion. Controls received Ringer's solution. IVM was performed 30‐100 min after reperfusion. Non‐perfused acini decreased and sinusoidal perfusion increased substantially after treatment. Leukocyte adherence to sinusoidal and venular endothelium was markedly reduced by C1‐INH. Transaminases were significantly decreased by C1‐INH. Our data obtained by IVM suggest that complement activation is an early key event of ischemia/reperfusion injury. These observations demonstrate for the first time that reperfusion related microcirculatory disorders can be minimized by C1‐INH. This compound should be evaluated in clinical application.     

NFκB抑制剂对白细胞介导肝脏缺血再灌注损伤的保护作用

目的　探讨大鼠肝脏缺血再灌注后核因子κB (NFκB)抑制剂对炎性介质表达和中性粒细胞浸润集聚的影响。方法　建立大鼠肝脏部分热缺血模型 ,实验组于缺血前 15min腹腔注射NFκB抑制剂脯氨酸二硫代氨基甲酸酯 (ProDTC 15mg/kg体重 ) ,对照组等量生理盐水注射。结果　再灌注 3h ,对照组NFκBP6 5达高峰 ,持续至 6h ;肿瘤坏死因子 α(TNF α)、巨噬细胞炎性蛋白 2(MIP 2 )、细胞间黏附分子 1(ICAM 1)mRNA表达和蛋白表达明显增强 ;再灌注 12h髓过氧化物酶(MPO)活性明显增高。应用ProDTC者 ,NFκBP6 5含量、TNF α、MIP 2、ICAM 1mRNA和蛋白表达量、MPO活性均降低 (P <0 0 5 )。再灌注 6hProDTC组也显著降低了天门冬氨酸转氨酶 (AST)、丙氨酸转氨酶 (ALT)、乳酸脱氢酶 (LDH)、W/D水平 ,和对照组相比差异显著 (P <0 0 5 )。结论　缺血再灌注后NFκB活化上调了炎性介质表达从而增加中性粒细胞浸润 ,通过ProDTC抑制NFκB活化可以减轻肝脏缺血再灌注损伤     

Rutin in rat liver ischemia/reperfusion injury: effect on DDAH/NOS pathway

Nitric oxide (NO) plays a key role in the relationship between microcirculatory disorders and I/R injuries. Our results demonstrated a significant modification in the hepatic function of I/R rats compared with the control group; treatment with rutin reported hepatic damage markers to control value. Levels of plasmatic and hepatic thiol groups decreased in the I/R untreated group, and this decrease was inhibited by rutin treatment. In addition, we observed an increase in the iNOS expression in I/R group compared with control and rutin administration attenuated this increase; in post-ischemic reperfused rutin-treated rats there was a significant increase in eNOS expression compared with the I/R untreated group. In the same experimental conditions an increase in DDAH 1 expression was observed in I/R group only; rutin treatment also counteracted this increased expression. These data suggest that rutin treatment could be useful for preventing oxidative damage associated with hepatic post-ischemic reperfusion injury.     

Hepatocyte protection by a protease inhibitor against ischemia/reperfusion injury of human liver

BACKGROUND: Total clamping of the hepatic pedicle can induce profound hepatic ischemia/reperfusion (I/R) injury, which remains a potentially lethal problem after hepatectomy. STUDY DESIGN: The purpose of this study was to evaluate the efficacy of a protease inhibitor in ameliorating I/R injury of the human liver. In a prospective, randomized, clinical study, 66 patients who underwent liver resection under conditions of continuous inflow occlusion were randomly assigned to three groups: 25 patients were given a synthetic protease inhibitor (gabexate mesilate [GM], 2.0 mg/kg/hr) intravenously starting 24 hours before surgery until postoperative day 3 (preop GM group); 16 were similarly given GM at the beginning of surgery (intraop GM group); and 25 served as controls (without GM group). Laboratory data and intraoperative and postoperative variables were analyzed and plasma levels of cytokines--tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6)--were measured to determine the relationship between surgical stress and hepatic I/R injury. RESULTS: The three groups of patients were similar in terms of age, gender, preoperative assessments, hepatic inflow occlusion time (approximately 50 minutes), extent of resection (proportion of major and minor hepatectomy), and background liver conditions. Preoperative administration of gabexate mesilate (preop GM group) substantially ameliorated hepatic I/R injury as compared with the other patients (intraop and without GM groups); postoperative serum transaminase levels were notably decreased in association with marked suppression of IL-6 levels in blood circulation during liver surgery. This was accompanied by a lower rate of postoperative complications and no mortality. Gabexate mesilate pretreatment abrogated the positive correlation between postreperfusion hepatocyte injury and hepatic ischemia time. CONCLUSIONS: Preoperative administration of GM is useful for preventing I/R injury of the human liver, accompanied by suppression of the plasma proinflammatory cytokine IL-6.     

Caspase 3 inhibition improves survival and reduces early graft injury after ischemia and reperfusion in rat liver transplantation

BACKGROUND: Apoptosis plays a crucial role after ischemia-reperfusion in organ transplantation. It is executed by caspases and influenced by the rheostat of pro- and anti-apoptotic proteins of the bcl-2 family. This study investigated the effect of specific inhibition of caspases 3 and 7 on graft function, survival, and hepatic bcl-2 levels after liver transplantation. METHODS: Lewis rats underwent syngeneic orthotopic liver transplantation after 16 hr of cold graft storage (in University of Wisconsin solution). Livers of donor animals treated with D(OMe)E(OMe)VD(OMe)-fluoromethylketone (specific inhibitor of apoptosis executor caspases 3 and 7), and appropriate control groups, were investigated. Early graft injury was quantified by measurement of bile flow and determination of microvascular graft injury by using in vivo fluorescence microscopy. Apoptosis and its regulation were examined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining and Western blot analysis of cell death effectors, respectively. RESULTS: After specific in vivo caspase inhibition, Western blot analysis revealed inhibition of caspase-induced cleavage of poly-ADP-ribose-polymerase. Inhibition of caspases 3 and 7 resulted in a significantly decreased number of apoptotic endothelial cells and improved microvascular perfusion. A cell protective effect was also suggested by an increase of bcl-2 levels at 7 days. Most important, specific caspase blockade resulted in improved rat survival after liver transplantation. CONCLUSION: Specific inhibition of apoptosis executor caspases effectively reduces graft ischemia-reperfusion injury and improves survival in liver transplantation. Better tissue preservation after caspase inhibition correlates with reduced apoptosis execution, improved microvascular perfusion, and bcl-2 up-regulation. Therefore, specific caspase inhibition represents a promising regimen for clinical use in liver transplantation.