Effect of ischemia-reperfusion injury on the microcirculation of the steatotic liver of the Zucker rat.

BACKGROUND: Much discussion has been focused on the use of steatotic livers for transplantation due to the prevalence of steatosis in the potential donor liver pool (1). The aim of this study was to investigate the possibility that the microcirculation of steatotic liver is more sensitive to the ischemia-reperfusion (IR) injury than normal liver. METHODS: The left liver lobe of obese (n=9) and lean Zucker rats (n=9) were subjected to 40 min of warm ischemia followed by 60 min of reperfusion. Fluorescent probes rhodamine 123 (Rh123), bisbenzimide (Bis), and rhodamine 6G (Rh6G) were administered for the identification by intravital fluorescence microscopy (IVFM) of mitochondrial membrane potential, hepatocyte nuclei and leukocytes, respectively before hepatic ischemia and at 15, 30, 45, and 60 min after reperfusion. Blood samples were obtained before and after 60 min of reperfusion. Liver tissue was taken at the end of experiment for histological analysis. RESULTS: The liver of the obese rats showed prominent macro- and microvesicular fatty changes (MAFC and MIFC) and hepatocyte swelling. Under IVFM, the obese animals had significantly wider hepatic cords (23.1+/-0.8 microm) than the lean ones (15.9+/-0.5 microm) (P<0.01), whereas no significant difference in sinusoidal diameters was noted. The number of functional sinusoids significantly decreased after 30 min of reperfusion in both groups but no significant change was noted in the nucleus count throughout the experiment. Rh123 fluorescence intensity dropped significantly in the obese group after 60 min of reperfusion but not in the lean rats. Leukocyte adherence showed a significant rise after reperfusion in both groups. Plasma AST and ALT levels were 40- and 24-fold higher respectively for the obese animals after IR compared with their preischemic values, whereas the corresponding increase were 4.2- and 3.4-fold for the lean animals, respectively. CONCLUSIONS: Our results indicate that the liver of the obese Zucker rat is steatotic and presents with an abnormal microcirculation manifested by a reduced sinusoidal density. IR led to significantly greater hepatic injury in the steatotic than in the normal liver. This injury was accompanied by a significant reduction in the functional sinusoidal density and mitochondrial membrane potential as assessed by Rh123-associated fluorescence in the steatotic liver. In conclusion, the increased sensitivity of the steatotic liver to IR injury would appear to involve both alterations in blood flow in the microcirculation and to cellular changes.     

Synergistic effects of brain death and liver steatosis on the hepatic microcirculation

BACKGROUND: The routine transplantation of steatotic livers could potentially mitigate the donor shortage, but so far is associated with a high rate of graft dysfunction. Steatosis and brain death have been perceived as independent risk factors, but they may synergistically target the hepatic microcirculation. This study compares the effects of brain death on the microcirculation of steatotic and normal livers. METHODS: Brain death was induced in obese and lean Zucker rats. Lean and obese sham-operated animals served as controls. Liver microcirculation was investigated using intravital fluorescence microscopy. Serum liver enzyme and reduced glutathione, expression of P-selectin, ICAM-1 and VCAM-1 mRNA in the liver were determined. The ultrastructural alterations were compared by electron microscopy. RESULTS: In nonbrain-dead animals, liver steatosis was associated with smaller sinusoidal diameters, but did not impair sinusoidal perfusion. During brain death, sinusoidal diameter and perfusion were reduced in normal and, to a greater extent, in steatotic livers. Also, more leukocytes were recruited to the microvasculature of steatotic livers than to normal livers in brain-dead state. The highest liver enzyme activities and the lowest hepatic GSH concentrations were measured in brain-dead animals with steatotic livers; only in these organs was endothelial cell swelling regularly observed. In brain-dead state, only the P-selectin mRNA expression was increased in steatotic livers as compared to normal livers. CONCLUSIONS: Brain death amplifies the adverse effects of steatosis on the hepatic microcirculation. Our results underline the need for therapeutic intervention in brain-dead state when steatotic livers are to be used for transplantation.     

热休克蛋白70和血红素加氧酶-1表达在肾缺血后处理减轻肾缺血再灌注损伤中的作用

目的 评价热休克蛋白70(HSP70)和血红素加氧酶-1(HO-1)表达在肾缺血后处理减轻肾缺血再灌注损伤中的作用.方法健康雄性SD大鼠140只,体重250～280 g,采用随机数字表法,将大鼠随机分为4组(n=35):假手术组(S组)仅开腹,游离双侧肾脏,分离双侧肾蒂不夹团;肾缺血再灌注组(I/R组)夹闭双侧肾蒂缺血45 min,恢复灌注;缺血后处理组(IPo组)夹闭双侧肾蒂45 min,再灌注10 s,缺血10 s,反复3次,恢复灌注;HSP抑制剂槲皮黄酮+缺血后处理组(Q+IPo组)缺血前1 h 腹腔注射槲皮黄酮100 mg/kg,余操作同IPo组.于再灌注即刻(T0)、1、3、6、12、24、48 h(T1～6)时各组随机取5只大鼠抽心脏血后取肾,检测肾组织HSP70、HO-1的mRNA和蛋白表达,T3时抽心脏血,测定血清肌酐(Cr)和尿素氮(BUN)浓度、caspase-3 mRNA的表达,TUNNEL法检测肾组织凋亡细胞,计算凋亡指数(AI),光镜下观察肾组织病理学结果.结果 与S组比较,其余组T3时血清Cr和BUN浓度和AJ升高,caspase-3 mRNA表达上调,各时点HSF70、BO-1的mRNA和蛋白表达上调(P＜0.05);与I/R组比较,IPo组T3时血清Cr和BUN浓度和AI降低,caspase-3 mRNA表达下调,T1～5时HSP70、HO-1的mRNA和蛋白表达上调(P＜0.05);与IPo组比较,Q+IPo组T3时血清Cr和BUN浓度和AJ升高,caspase-3mRNA表达上调,T1～5时HSP70、HO-1的mRNA和蛋白表达下调(P＜0.05).IPo组肾组织病理学损伤较I/R组减轻,Q+IPo组肾组织病理学损伤程度与I/R组相似.结论 HSP70和H0-1表达参与了肾缺血后处理减轻肾缺血再灌注损伤的过程.

Abstract：

Objective To evaluate the role of the expression of heat shock protein 70 (HSP70) and heme oxygenase-1 (HO-1) in the reduction of renal ischemia-reperfusion (I/R) injury by ischemic postconditioning in tats.Methods One hundred and forty healthy male SD rats weighing 250-280 g were randomized into 4 groups ( n = 35 each) : sham operation group (S group) ; I/R group; ischemic postconditioning group (IPo group); quercetin (an inhibitor of HSP) + ischemic postconditioning group (Q + IPo group). Renal I/R was produced by clamping bilateral renal pedicels for 45 min followed by reperfusion. In group S, bilateral kidneys were only exposed through a midline incision but their- pedicels were not clamped. In IPo and Q + IPo groups, 45 min ischemia was followed by three 10 s episodes of ischemia at 10 s intervals for reperfusion and in addition intraperitoneal quercetin 100 mg/kg was injected at 1 h before ischemia in group Q + IPo. Blood samples from hearts were obtained at 0, 1, 3, 6, 12, 24 and 48 h of reperfusion (T0-6) and the rats were then sacrificed and kidneys removed to detect the expression of HSP70 and HO-1 mRNA and protein in renal tissues. The blood samples obtained at T3 were used to determine serum creatinine (Cr) and urea nitrogen (BUN) concentrations and the expression of caspase-3 mRNA . The apoptosis in the renal tissues was detected using TUNEL and apoptotic index ( AI) was calculated. Microscopic examination was performed with light microscope. Results Compared with group S, the serum Cr and BUN concentrations and AI were significantly increased at T3,the expression of caspase-3 mRNA was up-regulated at T3, and the expression of HSP70 and HO-1 mRNA and protein was up-regulated at T0-6 in the other groups (P ＜ 0.05) . Compared with group I/R, the serum Cr and BUN concentrations and AI were significantly decreased at T3, the expression of caspase-3 mRNA was down-regulated at T3, and the expression of HSP70 and HO-1 mRNA and protein was up-regulated at T1-5 in group IPo ( P ＜ 0.05) . Compared with group IPo, the serum Cr and BUN concentrations and AI were significantly increased at T3, the expression of caspase-3 mRNA was up-regulated at T3, and the expression of HSP70 and HO-1 mRNA and protein was down-regulated at T1-5, in group Q + IPo ( P ＜ 0.05) . The microscopic examination showed that the renal I/R injury was significantly attenuated by ischemic postconditioning and the degree of injury in group IPo was similar to that in group I/R. Conclusion The expression of HSP70 and HO-1 is involved in the reduction of renal I/R injury by ischemic postconditioning in rats.     

缺血或药物预处理对大鼠供肝缺血再灌注损伤的抑制作用

目的　探讨缺血预处理 (IPC)或阿霉素预处理 (DPC ,模拟IPC)对大鼠供肝延迟性保护作用的发生机制。方法　将供鼠分为 3组。IPC组 :供鼠采用肝脏预先缺血 10min后再开放 ;DPC组 :供鼠经静脉注射阿霉素 (1mg/kg体重 ) ;对照组 :供鼠用等量生理盐水注射。观察各组预处理后血红素氧化酶 1(HO 1)和热休克蛋白 70 (HSP70 )含量 ;建立上述各组大鼠原位肝移植模型 ,并设假手术对照组 ,观察肝移植后各组对供肝缺血再灌注损伤的影响。结果　IPC组HO 1、HSP70含量分别于预处理 12h和 2 4h达到高峰 ;IPC和DPC组预处理 2 4h ,诱导的HSP70、HO 1含量差异无显著性 (P >0 .0 5 )。对照组肝移植后 6h ,肝组织中ICAM 1mRNA表达和内皮细胞ICAM 1分子表达明显增强 ,髓过氧化物酶 (MPO)活性增高 ,血清中天冬氨酸转氨酶 (AST)、丙氨酸转氨酶 (ALT)、乳酸脱氢酶 (LDH)及肝组织湿重 /干重 (W/D)水平明显升高 ,和假手术组相比 ,差异有显著性 (P <0 .0 1)。IPC或DPC组肝移植后减弱了ICAM 1mRNA和蛋白表达及MPO活性 ,AST、ALT、LDH及W/D的水平亦明显降低 ,与对照组比较 ,差异有显著性 (P <0 .0 5 )。结论　IPC的延迟保护作用是通过降低中性粒细胞的粘附浸润来实现的 ,这与IPC诱导生成HSP70和HO 1有关。DPC可以模拟IPC的延迟性保护     

The effect of methylprednisolone on warm ischemia-reperfusion injury in the liver

Background

Liver ischemia-reperfusion (I-R) injury is a well-known cause of morbidity and mortality following liver surgery and transplantation. Further investigation is warranted to identify measures that reduce the untoward sequelae of liver ischemia.

Methods

Male Sprague-Dawley rats (wild-type) and Zucker rats (with hepatic steatosis) were subjected to 75 minutes of 70% hepatic ischemia and 3 hours of reperfusion. The ischemic periods were based on protocols of either continuous clamping (CC) or ischemic preconditioning (IP). Prior to ischemia induction, rats were pretreated with intravenous methylprednisolone (MP; 2 mg/kg) or normal saline. Warm I-R injury was evaluated using serum levels of aspartate aminotransferase (AST), serum interleukin-6 (IL-6), and hematoxylin and eosin staining.

Results

Histology, serum IL-6, and AST release revealed that MP treatment provided significant protection as compared with ischemic controls (both CC and IP groups) only in the normal, not steatotic, livers. The inflammatory response was considerably reduced in MP groups with normal livers but not in steatotic livers. In general, the IP groups showed decreased I-R injury compared to the CC group. However, MP was able to further reduce I-R injury only in normal, not steatotic, livers.

Conclusions

MP attenuated the postischemic and inflammatory response in the normal, and not steatotic, livers. MP pretreatment might be effective in reducing warm I-R injury to livers without steatosis. The mechanism of I-R-related hepatocellular damage in steatotic liver is different than in normal liver.     

Silent information regulator 1 protects the liver against ischemia–reperfusion injury: implications in steatotic liver ischemic preconditioning

Ischemia–reperfusion (IR) injury is an important problem in liver surgery especially when steatosis is present. Ischemic preconditioning (PC) is the only surgical strategy that has been applied in patients with steatotic livers undergoing warm ischemia. Silent information regulator 1 (SIRT1) is a histone deacetylase that regulates various cellular processes. This study evaluates the SIRT1 implication in PC in fatty livers. Homozygous (Ob) Zucker rats were subjected to IR and IR + PC. An additional group treated with sirtinol or EX527 (SIRT1 inhibitors) before PC was also realized. Liver injury and oxidative stress were evaluated. SIRT1 protein levels and activity, as well as other parameters involved in PC protective mechanisms (adenosine monophosphate protein kinase, eNOS, HSP70, MAP kinases, apoptosis), were also measured. We demonstrated that the protective effect of PC was due in part to SIRT1 induction, as SIRT1 inhibition resulted in increased liver injury and abolished the beneficial mechanisms of PC. In this study, we report for the first time that SIRT1 is involved in the protective mechanisms induced by hepatic PC in steatotic livers.     

Efficacy of HSP72 induction in rat liver by orally administered geranylgeranylacetone

Abstract It is well known that heat‐shock proteins (HSPs) have a cyto‐protective function as “molecular chaperones” when cells are exposed to several stress conditions. Geranylgeranylacetone (GGA) is an antiulcer drug that was developed in Japan and it has recently been reported to induce HSP72 in rat gastric mucosa. In this experiment, we investigated the induction of HSP72 in rat liver in response to oral administration of GGA and assessed its ability to induce tolerance to warm ischemic injury by this approach. We prepared donor rats by orally administering GGA to them and compared HSP72 expression in graft liver, survival rates, and serum TNF‐α concentrations after liver transplantation with the findings in controls. The survival rates were significantly increased when the livers were obtained from donor rats given GGA. Western blotting revealed expression of HSP72 in graft livers given GGA, and the serum TNF‐α levels were significantly suppressed in the rats given GGA. Oral administration of GGA induced HSP72 in graft livers, and they were better able to tolerate warm ischemic injury. Oral administration of GGA appears to provide a promising new strategy for preventing ischemia‐reperfusion injury.     

Value of α glutathione S‐transferase for in vitro evaluation of preservation injury in normal and steatotic livers

Abstract Liver steatosis is frequently encountered at organ harvest and, although functionally in‐apparent in the donor, may seriously affect the functional recovery of the graft after ischemic preservation. The present study was aimed to investigate the diagnostic value of alpha‐glutathione S‐transferase (GST) in non‐schemic and ischemic livers with or without compensated steatosis. A histologically documented mild to moderate steatosis was induced in livers of male Wistar rats by fasting for 2 days and subsequent feeding of a fat‐free diet enriched in carbohydrates. Fatty livers (FL) were retrieved and perfused in vitro for 45 min either immediately or after ischemic preservation at 4°C in HTK solution. Effluate was collected during isolated perfusion and later analysed for liver specific enzymes, including GST. Normal livers (NL) were excised from healthy rats and underwent the same protocol. Non‐ischemic livers showed similar enzyme release (FL versus NL) for ALT or GLDH but significant differences in GST. After ischemic preservation of NL, enzyme release increased mildly with respect to the non‐ischemic reference values for ALT, remained unchanged for GLDH and rose substantially for GST. In FL, there was a more than 10‐fold increase in all parameters, being most pronounced for GLDH as a marker of mitochondrial damage. It is concluded that GST may discriminate between healthy and suboptimal steatotic livers prior to ischemia and that the release of GST upon postischemic reperfusion of normal livers proves to be the most sensitive indicator for hepatocellular injury. However, GST turned out to be less useful for the detection of postischemic reperfusion injury in steatotic grafts.     

Ischemia–reperfusion injury in patients with fatty liver and the clinical impact of steatotic liver on hepatic surgery

Hepatic steatosis is one of the most common hepatic disorders in developed countries. The epidemic of obesity in developed countries has increased with its attendant complications, including metabolic syndrome and non-alcoholic fatty liver disease. Steatotic livers are particularly vulnerable to ischemia/reperfusion injury, resulting in an increased risk of postoperative morbidity and mortality after liver surgery, including liver transplantation. There is growing understanding of the molecular and cellular mechanisms and therapeutic approaches for treating ischemia/reperfusion injury in patients with steatotic livers. This review discusses the mechanisms underlying the susceptibility of steatotic livers to ischemia/reperfusion injuries, such as mitochondrial dysfunction and signal transduction alterations, and summarizes the clinical impact of steatotic livers in the setting of hepatic resection and liver transplantation. This review also describes potential therapeutic approaches, such as ischemic and pharmacological preconditioning, to prevent ischemia/reperfusion injury in patients with steatotic livers. Other approaches, including machine perfusion, are also under clinical investigation; however, many pharmacological approaches developed through basic research are not yet suitable for clinical application.     

Induction of Heat Shock Response: Effect on the Rat Liver with Carbon Tetrachloride-induced Fibrosis from Ischemia-reperfusion Injury

n = 56) received no pretreatment except anesthesia, and the heat shock group (group HS, n = 56) were exposed to heat shock (42°C) for 15 minutes. After a 48-hour recovery all rats were subjected to 30 minutes of warm ischemia. Western blotting analysis was employed for heat shock protein (HSP) 72 detection. The adenine nucleotide levels in liver tissue and the liver enzyme levels in serum were measured before and after ischemic intervention (seven animals were used at each of six time point measurements in both groups). HSP72 was induced in group HS at greater intensity than in group C. The survival rate on postoperative day 7 in group C (3/14) was significantly poorer than that in group HS (14/14) ( p < 0.01). The higher survival rate in group HS was accompanied by more rapid recovery of the adenosine triphosphate level and lower serum levels of liver enzymes after reperfusion ( p < 0.01 vs. group C). Heat shock preconditioning induces HSP72 in the rat liver with fibrosis and provides significantly increased tolerance of warm-ischemia reperfusion injury.     

Contributions of heme oxygenase-1 in postconditioning-protected ischemia-reperfusion injury in rat liver transplantation

Background

Heme oxygenase-1 (HO-1), an oxidative stress-response gene up-regulated by various physiological and exogenous stimuli, has cytoprotective activities. Ischemic postconditioning (Postcon) can protect an organ from ischemia-reperfusion (I/R) injury. In the present study, we investigated the potential contributions of HO-1 to Postcon-dependent protection against I/R injury in rat liver transplantation models.

Materials and methods

Adult male Sprague-Dawley rats were randomly divided into four groups: sham group with laparotomy for liver exposure; I/R group with 24-hour cold ischemia of the donor liver; Postcon group with the same treatment as the I/R group plus ischemic Postcon; and zinc protoporphyrin (ZnPP HO-1 inhibitor) + Postcon group treated the same as the Postcon cohort with donors pretreated using ZnPP 24 hours before the I/R injury. We measured liver tissue and peripheral blood samples collected at 6 hours after reperfusion and serum transaminase levels, histopathology, liver tissue malondialdehyde (MDA) content, superoxide dismutase (SOD) activity and HO-1 expression in the liver.

Results

Postcon significantly diminished the elevation of serum transaminases levels after I/R injury when compared with I/R and ZnPP+Postcon groups. Postcon treated rats showed significantly lower MDA production and higher SOD activity. HO-1 was induced in rat livers exposed to Postcon; its levels were obviously overexpressed after 6 hours in Postcon rats. Inhibiting the expression of HO-1, negated the protective effects of Postcon.

Conclusions

Induction of HO-1 in the Postcon condition played a protective role against hepatic I/R injury and enhanced the early antioxidative activity. The protective effects of Postcon were significantly associated with greater intrahepatic HO-1 expression.     

Ergothioneine pretreatment protects the liver from ischemia-reperfusion injury caused by increasing hepatic heat shock protein 70

BACKGROUND: Reperfusion of the liver after ischemia induces the expression of the heat shock genes and the synthesis of the heat shock proteins (HSP). We studied the effects of the natural antioxidant ergothioneine (EGT) treatment on the expression of HSP70 in ischemic-reperfused (IR) liver. METHODS: Adult male Wistar rats were randomly divided into three groups: Sham group given standard laboratory chow and water for 3 weeks followed by sham operation; Control group given standard laboratory chow and water for 3 weeks followed by liver IR injury; EGT group given standard laboratory chow supplementation l-ergothioneine (1.2 mg/kg/d body weight) administered by gavage and water for 3 weeks followed by liver IR injury. Ten rats from each group were killed to determine serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic dehydrogenase (LDH), tissue malondialdehyde (MDA), HSP70 levels, and histologic changes at 30, 60, and 120 min of reperfusion, respectively. Survival was followed for 1 week. RESULTS: IR caused significant increase in serum AST, ALT, LDH, and tissue MDA levels. As compared with the control group, animals treated with EGT experienced a significant decrease in serum AST, ALT, and LDH levels in all reperfusion periods. Tissue MDA levels in animals receiving EGT were significantly reduced as compared with control group at 30 min and 60 min after reperfusion. After ischemia, reperfusion caused a remarkable production of HSP70 in the control group. When the rats were pretreated with EGT, the levels of HSP70 increased significantly in their livers after reperfusion compared with the control group. Liver injury in the EGT-treated animals was lower to that in the control group. The 7-day survival rate was significantly improved (from 50% to 80%) by EGT pretreatment. CONCLUSION: HSP70 has been shown to induce tolerance against warm IR injury in rat livers. EGT pretreatment protects the liver from IR injury by over-expression of HSP and the subsequent suppression of lipid peroxidation.     

Vitamin E Succinate Enhances Steatotic Liver Energy Status and Prevents Oxidative Damage Following Ischemia/Reperfusion

We have previously shown that treatment of steatotic livers with vitamin E succinate decreases liver injury and increases survival after ischemia/reperfusion (I/R). It is now understood that compromised energy status is associated with increased injury following liver ischemia in the setting of hepatic steatosis at least partially as a result of increased reactive oxygen species (ROS) and induction of mitochondrial uncoupling protein-2 (UCP2). Given the association between ROS, mitochondrial function, and UCP2, it was our goal to determine whether the protective effects of vitamin E succinate were associated with decreased ROS injury, down-regulation of UCP2, or improvement of ATP levels following I/R. To test this, leptin deficient (ob/ob) mice with steatotic livers that had received other 50 IU of vitamin E succinate supplement per day or control chow for 7 days were subjected to total hepatic ischemia (15 minutes) followed by reperfusion. We measured liver expressions of ATP, glutathione (GSH), and UCP2 as well as mitochondrial DNA damage. Vitamin E treatment decreased hepatic UCP2 expression and increased ATP and GSH levels prior to I/R. These levels were maintained at 1 hour after I/R. At 24 hours, while hepatic UCP2 expression, ATP, and GSH levels were similar to those of mice not receiving vitamin E, mitochondrial DNA damage was blocked. These results revealed that vitamin E succinate decreased hepatic UCP2 expression, reduced oxidative stress, and improved mitochondrial function in mice with steatotic livers before and after I/R, identifying mechanisms of protection in this setting.     

Ageing and Hepatic Steatosis Exacerbate Ischemia/Reperfusion-Accelerated Outgrowth of Colorectal Micrometastases

Background Ischemia/reperfusion (I/R) injury is frequently encountered during hepatic surgery. We recently showed that I/R accelerates the outgrowth of pre-established colorectal micrometastases. The aim of this study was to assess the influence of ischemia time, gender, age, and liver steatosis on the accelerated outgrowth of colorectal metastases following I/R. Methods Five days after tumor cell inoculation, mice were subjected to 20, 30 or 45 min of left lobar I/R. To assess the influence of age, gender, and liver steatosis on I/R-accelerated tumor growth, we compared old with young mice, male with female mice, and mice with healthy livers with mice with steatotic livers. Endpoints were extent of tissue necrosis and tumor growth. Results With increasing ischemia times, tissue necrosis and I/R-accelerated tumor growth increased, with a significant stimulatory effect at 30 and 45 min of ischemia. I/R-stimulated outgrowth of micrometastases was further increased by 33% in aged mice and by 42% in steatotic livers and was associated with increased tissue necrosis. In female mice tissue necrosis had decreased by 47% and tumor growth was reduced in both control and clamped liver lobes. The stimulatory effect of I/R on metastasis outgrowth was similar in male and female mice. Conclusions I/R-accelerated outgrowth of colorectal micrometastases largely depends on the duration of the ischemic period, with a safe upper limit of 20 min in mice. The stimulatory effects of I/R on tumor growth are exacerbated in aged mice and in steatotic livers.     

Protective effect of sivelestat,a neutrophil elastase inhibitor,on ipsilateral and contralateral testes after unilateral testicular ischaemia–reperfusion injury in rats

What’s known on the subject? and What does the study add? Following ischemic damage, reperfusion may cause further injury paradoxically in the ischemic tissue, known as reperfusion injury. Decreased blood flow causes hypoxia, leading to increased levels of lactic acid, hypoxanthine, and lipid peroxides in ischemic tissues and subsequent increase in blood flow after lipid peroxidation produces reactive oxygen species. In addition, several experimental studies and clinical trials demonstrated that unilateral testicular torsion has a detrimental effect also to the contralateral testis. Although the basic pathological mechanism underlying testicular ischemia/reperfusion injury has not been completely understood, it has been shown that reactive oxygen species formed during ischemia/reperfusion play the key role in this process. In the international literature there is no information available regarding the effects of neutrophil elastase inhibitors such as sivelestat sodium aminoacetate tetrahydrate on the ischemia/reperfusion injury of the testis. In this study we investigated the effects of sivelestat in the testes bilaterally, after unilateral testicular ischemia/reperfusion injury using an experimental unilateral testicular ischemia/reperfusion rat model. We found that sivelestat reduces the oxidative stress and partially prevents the testicular damage both in the ischemic and in the contralateral testis.

OBJECTIVE

To investigate the effect of a neutrophil elastase inhibitor, sivelestat sodium hydrate, on testicular ischaemia–reperfusion (IR)‐injury.

MATERIAL AND METHODS

Eight‐week‐old male Sprague–Dawley rats were divided into four groups: sham‐operated control rats; IR rats (group IR); and IR rats that received intra‐abdominal administration of 15 mg/kg or 60 mg/kg sivelestat (group IR15 and group IR60, respectively). Right testicular vessels were clamped for 90 min in groups IR, IR15 and IR60. Sivelestat had been administered 45 min after the induction of the ischaemia in groups IR15 and IR60. In subpopulations of IR, IR15 and IR60 rats, reperfusion was performed after ischaemia for 2 h (groups IR‐A, IR15‐A and IR60‐A, respectively) or 48 h (groups IR‐B, IR15‐B and IR60‐B, respectively). At the end of the reperfusion period, blood samples were aspirated from both spermatic veins of each rat and testosterone was evaluated. Then both testes from all rats were collected and tissue levels of malondialdehyde (MDA), myeloperoxidase (MPO), and heat‐shock protein‐70(HSP‐70) were evaluated. Testicular tissue samples were also processed for histological evaluation and TUNEL staining.

RESULTS

MDA, MPO and HSP‐70 levels in the ischemic testis were significantly higher in the IR group compared with the control group. MDA and HSP‐70 in the contralateral testis were significantly higher in the IR group compared with the control group. Bilateral testosterone levels were lower in all rat groups in comparison with the control group. Bilateral testicular samples in group IR showed extensive histopathologic degenerative alterations and increased percentage of apoptotic cells. Sivelestat treatment lowered the MDA concentration and the percentage of apoptotic cells bilaterally and ameliorated the testicular histological pattern bilaterally.

CONCLUSIONS

Unilateral testicular ischaemia causes significant contralateral testicular damage. Sivelestat may be a novel adjunct tool for reducing oxidative stress and partially preventing bilateral testicular damage.     

Transient limb ischemia induces remote preconditioning in liver among rats: the protective role of heme oxygenase-1

BACKGROUND: We have reported the protective role of heme oxygenase-1 (HO-1) in the mechanism of hypoxic preconditioning. We wish to investigate the role of HO-1 in remote preconditioning (RP) against hepatic ischemia/reperfusion (I/R) injury in rats. METHODS: The remote preconditioning was produced by four cycles of 10-min ischemia-reperfusion of the hind limb of rats. Partial hepatic ischemia was produced in the left lobes for 45 min followed by 240 min of reperfusion. Zinc-protoporphyrin IX (ZnPP), a specific inhibitor of HO enzymatic activity, was intra-peritoneally injected 1 hr before the ischemia-reperfusion injury in separate groups of RP rats. Serum alanine transaminase (ALT) levels, expression of hepatic HO-1 protein and mRNA, immunohistochemical staining and HO enzymatic activity were measured. RESULTS: HO-1 was induced in the livers of rats 4 hr after the RP stimuli, and the overexpression persisted for 24 hr. Immunohistochemical staining demonstrated induction of HO-1 in the hepatocytes. The peripheral lymphocytes did not express HO-1 after RP. RP diminished the elevation of serum ALT levels 4 hr after I/R injury (283.7+/-167.4 U L) when compared with controls (1297.7+/-729.3 U L) and RP+ ZnPP pretreated groups (1429.9+/-750.9 U L). The heme oxygenase activity in treated rats also correlated these results (286.8+/-34.3 pmol mg protein hr for the RP group, 156.3+/-27.5 pmol mg protein hr for the RP+ ZnPP pretreated group, and 170.6+/-19.4 pmol mg protein hr for the control group, 144.8+/-7.8 pmol mg protein hr for the control+ ZnPP pretreated group). CONCLUSION: Our results indicated that the induction of HO-1 in remote preconditioning played a protective role against hepatic I/R injury.     

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.     

The protective role of heme oxygenase-1 on the liver after hypoxic preconditioning in rats

BACKGROUND: Hypoxic preconditioning (HP) confers cytoprotection against ischemia/reperfusion (I/R) injury. This effect is in part attributable to the induction of heme oxygenase (HO)-1. This experiment evaluates liver cell damage after I/R injury in HP rats. METHODS: HP rats were prepared by exposure (15 hr/day) to an altitude chamber (5500 m) for 2 weeks. Partial hepatic ischemia was produced in the left lobes for 45 min followed by 180 min of reperfusion. Zinc (Zn) protoporphyrin (PP), a specific inhibitor of HO enzymatic activity, was subcutaneously injected 1 hr before the I/R injury into separate groups of sea-level (SL) control and HP rats. Serum alanine aminotransferase (ALT) levels, liver HO-1 mRNA and protein, and HO enzymatic activity were measured. RESULTS: HO-1 was induced in the livers of rats exposed to HP. The levels of HO-1 mRNA and protein were obviously overexpressed after 2 weeks of HP. HP diminished the elevation of serum ALT levels after I/R injury (83.7+/- 4.9 U/L) when compared with SL controls (280.8+/-19.4 U/L) and HP+ZnPP-pretreated groups (151.3+/-4.4 U/L). The HO activity in treated rats also was correlated with these results (237.9+/-19.8 pmol/mg of protein per hour for the HP group, 164.3+/-12.7 pmol/mg of protein per hour for the HP+ZnPP group, and 182.6+/-8 pmol/mg of protein per hour for the SL controls). CONCLUSIONS: The authors' results indicated that the induction of HO-1 in hypoxic preconditioning played a protective role against hepatic I/R injury.     

Cyclic RGD peptides with high affinity for alpha5beta1 integrin protect genetically fat Zucker rat livers from cold ischemia/reperfusion injury

We tested a hypothesis that interactions between fibronectin (FN), a key extracellular matrix component, and its integrin alpha5beta1 receptor are important in the development of ischemia/reperfusion (I/R) injury of steatotic liver transplants. We examined the effect of a cyclic RGD peptide (cRGD), with high affinity for alpha5beta1 integrin, in a well-established steatotic rat liver model of ex vivo cold ischemia followed by isotransplantation. In this model, cRGD peptides were administered through the portal vein of steatotic Zucker rat livers prior to and after cold ischemic storage. Lean Zucker recipients of fatty orthotopic liver transplants (OLTs) received an additional course of cRGD peptides 1 hour posttransplantation. cRGD peptide therapy significantly inhibited the recruitment of monocyte/macrophages, and repressed the expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Moreover, it resulted in selective inhibition of inducible nitric oxide synthase (iNOS), and MMP-9 expression. Importantly, cRGD peptide therapy improved the function and histologic preservation of steatotic liver grafts, extending their 14-day survival in lean recipients from 50% in untreated to 100% in cRGD-treated OLTs. Thus, cRGD peptide-mediated blockade of FN-alpha5beta1 interaction protects against severe I/R injury otherwise experienced by steatotic OLTs.     

Hepatic microcirculatory failure

Liver ischemia has been considered a frequent problem in medical practice, and can be associated to a number of surgical and clinical situations, such as massive hepatic resections, sepsis, liver trauma, circulatory shock and liver transplantation. After restoring blood flow, the liver is further subjected to an additional injury more severe than that induced by ischemia. On account of the complexity of mechanisms related to pathophysiology of ischemia and reperfusion (I/R) injury, this review deals with I/R effects on sinusoidal microcirculation, especially when steatosis is present. Alterations in hepatic microcirculation are pointed as a main factor to explain lower tolerance of fatty liver to ischemia-reperfusion insult. The employment of therapeutic strategies that interfere directly with vasoactive mediators (nitric oxide and endothelins) acting on the sinusoidal perfusion seem to be determinant for the protection of the liver parenchyma against I/R. These approaches could be very suitable to take advantage of marginal specimens as fatty livers, in which the microcirculatory disarrangements hamper its employment in liver transplantation.