Recombinant human soluble thrombomodulin (ART-123) prevents warm ischemia-reperfusion injury in liver grafts from non-heart-beating donors

Background

Liver transplantation is an established treatment for end-stage liver disease. However, an ongoing problem worldwide concerning this treatment is the shortage of grafts. Although transplantation using grafts from non-heart-beating donors (NHBDs) is considered a promising solution, some researchers have reported that these liver grafts are associated with primary graft nonfunction and biliary complications. The purpose of this study was to establish a safe technique procuring liver grafts from marginal donors such as NHBDs.

Materials and methods

Male Wistar rats were divided into three groups: (1) the heart-beating (HB) group, whose livers were retrieved from HB donors; (2) the non-HB (NHB) group, whose livers were retrieved from NHBDs that had experienced an apnea-induced agonal condition (for this group, livers were subjected to warm ischemia for 30 minutes after cardiac arrest); and (3) the recombinant human soluble thrombomodulin (ART-123) group, whose livers were retrieved in the same manner as the NHB group but pretreated with ART-123 (1 mg/kg) at the agonal stage. The livers were reperfused for 60 minutes with oxygenated Krebs-Henseleit bicarbonate buffer after cold preservation for 6 hours.

Results

Bile production and portal flow volume in the ART-123 group were significantly higher than those in the NHB group. Alanine aminotransferase levels in the ART-123 group were significantly lower than those in the NHB group. Histological findings showed the narrowing of sinusoidal spaces and necroses in the NHB group were more severe than those in the ART-123 group.

Conclusions

These results suggest that thrombomodulin may improve the viability of liver grafts from NHBDs.     

Perfusion using oxygenated buffer containing prostaglandin E1 before cold preservation prevents warm ischemia-reperfusion injury in liver grafts from non-heart-beating donors

We have previously reported that perfusion using warm oxygenated buffer before cold preservation (preperfusion) improved the viability of liver grafts from non-heart-beating donors. We demonstrated that adenosine triphosphate content was restored and apoptosis was reduced. The objective of the present study was to evaluate mitochondrial functions after this preperfusion and the effects of addition of prostaglandin E₁ (PGE₁) to the preperfusion buffer. Preperfusion improved portal flow, bile production, and mitochondrial function, and reduced alanine aminotransferase levels in the perfusate. Addition of PGE₁ significantly increased bile production and suppressed alanine aminotransferase and tumor necrosis factor-α levels. PGE₁ minimized mitochondrial membrane damage and ischemic injury after liver graft reperfusion. Release of mitochondrial cytochrome c was suppressed by addition of PGE₁. In conclusion, perfusion using oxygenated buffer containing PGE₁ before cold preservation significantly prevented cellular damage, protected mitochondrial function, and suppressed the release of mitochondrial cytochrome c in livers undergoing warm ischemia-reperfusion injury. This method shows promise for reducing cellular damage in non-heart-beating donor liver grafts.     

A new free radical scavenger,edaravone, ameliorates oxidative liver damage due to ischemia-reperfusion in vitro and in vivo

Ischemia-reperfusion injury causes oxidative stress producing reactive oxygen species, which is a serious problem linked to morbidity and mortality in liver surgery. We investigated the effects of edaravone, a new free radical scavenger, on liver oxidative stress in vitro and in vivo. We employed a hypoxiareoxygenation model of primary cultured hepatocytes using an AnaeroPack (Mitsubishi Gas Chemical Co., Tokyo, Japan). Hepatocytes were exposed to 3 or 4 hours of hypoxia and then returned to oxygenation. We analyzed the time course changes of aspartate aminotransferase (AST), phosphatidylcholine hydroperoxide (PCOOH), and adenosine triphosphate (ATP) content in hepatocytes of edaravone-treated groups or nontreated groups after reoxygenation. Edaravone significantly attenuated the elevation of the AST level of the medium and hepatocellular PCOOH and preserved the hepatocellular ATP level. In vivo, male Sprague-Dawley rats were subjected to 45 minutes of hepatic ischemia and 120 minutes of reperfusion. The rats were intravenously injected with vehicle or edaravone (3 mg/kg or 10 mg/kg) before reperfusion and 1 hour after reperfusion. Serum AST levels and hepatic PCOOH and energy charge were significantly improved in both edaravone groups compared with control. In conclusion, edaravone has the ability to eliminate intra-hepatocellular superoxide species and attenuate oxidative liver damage in liver surgery. This work was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan and the Yamanouchi Foundation.     

Tempol, an intracelullar free radical scavenger, reduces liver injury in hepatic ischemia-reperfusion in the rat

Liver ischemia is of clinical interest because of its role in liver failure and also hepatic graft rejection. The generation of reactive oxygen species contributes to the injury that follows ischemia-reperfusion. One therapy utilizes the administration of antioxidants; however, only limited experience suggests a potential benefit of systemic administration of these compounds. To overcome the limitations of these compounds, small molecules with improved cell membrane permeability characteristics and higher potency, such as tempol, are being tested in vivo. Tempol, a membrane-permeable radical scavenger, interferes with the formation or the effects of many radicals, including superoxide anions, hydroxyl radicals, and peroxynitrite. The aim of this study was to investigate the effects of tempol in an in vivo rat model of liver ischemia-reperfusion injury. Male Wistar rats were pretreated with tempol (30 mg/kg, i.v.) 5 minutes prior to liver ischemia (for 30 minutes) and reperfusion (for 2 hours). The liver injury was assessed by measuring serum levels of transaminases, lactate dehydrogenase, and gamma-glutamyl transferase. Tempol significantly mitigated the increase in transaminases, lactate dehydrogenase, and gamma-glutamyl transferase following liver ischemia-reperfusion, suggesting an improvement in liver function and resistance to injury.     

A radical scavenger, edaravone, protects canine kidneys from ischemia-reperfusion injury after 72 hours of cold preservation and autotransplantation

BACKGROUND: Cold ischemia-reperfusion (I/R) injury is a prominent cause of delayed graft function after kidney transplantation. Reactive oxygen species play a crucial role in I/R injury. Edaravone is a synthetic radical scavenger that has been used in acute stroke. Some animal experiments have revealed its beneficial effects against I/R injury, but its effects after cold preservation and transplantation of canine kidneys are unknown. METHODS: Female hybrid dogs weighing 11 to 13 kg were used. Under anesthesia, the left kidney was harvested. After 72 hr of preservation in cold histidine-tryptophan-ketoglutarate solution, autotransplantation was performed in the right iliac fossa, with contralateral nephrectomy. Animals were divided into control and treatment groups (n=6 per group). In the treatment group, edaravone was administered intravenously at harvest and at reperfusion (3 mg/kg) and in addition was added to the preservation solution (50 microM). RESULTS: Animal survival at 2 weeks was four of six in the control group and six of six in the treatment group. Compared with controls, treated animals had higher mean urine output, higher mean glomerular filtration rate, improved tubular cell function, lower mean serum creatinine, and lower renal vascular resistance. Biopsy specimens from treated animals showed less tubular cell damage and decreased P-selectin expression in endothelial cells. Lipid peroxidation of renal tissue and urinary excretion of 8-hydroxy-2'-deoxyguanosine were suppressed by the treatment. CONCLUSIONS: Edaravone reduced cold I/R injury in canine renal transplantation. The agent has the potential to ameliorate preservation injury in clinical transplantation.     

Kidney transplantation with grafts from non-heart-beating donors

We analyzed the function and outcome of 16 kidney transplants performed in our hospital from non-heart-beating donors who were harvested at other hospitals. The cold ischemia times were longer and the delayed graft function rates higher. However, graft function was no different from that of kidneys from heart-beating donors. This experience has encouraged us to use this type of donor to reduce the transplant waiting list.     

Evaluation of ischemic injury during liver procurement from non-heart-beating donors

The aim of this study was to assess liver viability after different periods of cardiac arrest and the predictive value of two markers of ischemia-reperfusion injury. METHODS: A pig liver transplantation model of non-heart-beating donors was studied. Four donor groups were designed; three groups were submitted to different periods of cardiac arrest (20, 30 and 40 min), and the fourth group served as the control group (without cardiac arrest). In the non-heart-beating donor groups, normothermic recirculation was established 30 min prior to total body cooling. Aminotransferase, alpha-glutathione-S-transferase, and hyaluronic acid determinations as well as liver biopsies, were serially performed. RESULTS: Although hepatocellular function could be preserved after 40 min of cardiac arrest, histological lesions at 5 days were considered irreversible due to the presence of a necrotic biliary tract. An overall significant relationship was found between the time period of cardiac arrest (20, 30 or 40 min) and the levels of hyaluronic acid (p = 0.004) or alpha-glutathione-S-transferase (p = 0.01) obtained during liver procurement and transplantation. CONCLUSIONS: The period of cardiac arrest is the determinant factor of liver viability after liver transplantation from non-heart-beating donors. As early markers of endothelial or hepatocellular damage, hyaluronic acid or alpha-glutathione-S-transferase levels may help to evaluate the ischemic injury of a potential donor.     

Maximizing liver transplantation from non-heart-beating donors

In order to increase the supply of transplantable organs, an increasing number of organ procurement organizations are adopting policies regarding donations from non-heart-beating donors. Few centers, however, actually recover and transplant these organs. This article reviews a case in which kidneys and livers imported from out of state were successfully recovered from a non-heart-beating donor and transplanted. In addition, the article demonstrates how cooperation and flexibility in transplant personnel can increase the number of organs transplanted from a non-heart-beating donor.     

Intrathecal edaravone, a free radical scavenger, is effective on inflammatory-induced pain in rats

BACKGROUND: Free radicals have some roles in inflammation and systemic and local tissue injuries. (Free radical scavengers are neuroprotective against excitotoxic insults.) Therefore, we hypothesized that free radical scavenger would be analgesic on pain induced by excitotoxicity or inflammation. The purpose of this study was to investigate analgesic effects of intrathecally administered edaravone, a free radical scavenger, on thermal and inflammatory pain. METHODS: Sprague-Dawley rats were implanted with lumbar intrathecal catheters. Edaravone 0.05, 0.1, 0.5, and 1 mg per 20 microl or saline 20 microl (control) were administered intrathecally, and the withdrawal response to thermal stimulation to the tail (tail-flick test) or flinch responses to subcutaneous formalin injection into the hind paw (formalin test) were tested. General behaviour and motor function were also examined. In each dose group, eight rats were used. RESULTS: No dose-dependent analgesic effects were observed in the tail-flick test. However, dose-dependent analgesia was obtained in both phase 1 and 2 of the formalin test. The 50% effective dose values were 0.25 mg (95% confidence interval, 0.11-0.56 mg) in phase 1 and 0.25 mg (95% confidence interval, 0.061-1.05 mg) in phase 2. No behavioural side-effects nor motor dysfunction was observed, even with the maximum soluble dose (1 mg/20 microl). CONCLUSION: Intrathecally administered edaravone, a free radical scavenger, had analgesic effects on inflammatory-induced acute and facilitated pain but not on acute thermal pain, without any behavioural side-effects.     

Elimination of Kupffer cells and nafamostat mesilate rinse prevent reperfusion injury in liver grafts from agonal non-heart-beating donors.

BACKGROUND: We hypothesized that microcirculatory disturbance was an obstacle to liver transplantation (LTx) from non-heart-beating donors (NHBDs) and that it was attributed mainly to a deterioration of sinusoidal endothelial cells (SECs) and sinusoidal narrowing. This study was designed to examine porcine orthotopic LTx using livers obtained from pretreated agonal NHBDs, and to determine whether the maintenance of the liver microcirculation would result in successful LTx from agonal NHBDs. METHODS: Pigs were allocated to five groups: (i) control group; (ii) NM group, in which grafts were rinsed with nafamostat mesilate (NM) rinse; (iii) LD group, in which Kupffer cells in grafts were eliminated by liposome-encapsulated dichloromethylene diphosphonate (L-DMDP); (iv) LDNM group, in which grafts pretreated with L-DMDP were rinsed with NM rinse; (v) heart-beating donor (HBD) group. In all groups, but the HBD group, the livers were pretreated with FK506 and prostaglandin I2 analogue, and were preserved in University of Wisconsin solution after cardiac arrest. Thereafter orthotopic LTx was performed. RESULTS: After reperfusion, it was histologically demonstrated that elimination of Kupffer cells prevented SECs deterioration and NM rinse prevented sinusoidal narrowing. The hepatic energy charge recovered in all groups except the control group. In the LDNM group, three of four recipients survived more than 7 days. CONCLUSIONS: For a successful LTx from agonal NHBDs, it is important to prevent microcirculatory disturbance caused by SEC deterioration and sinusoidal narrowing after reperfusion. Combination therapy consisting in the elimination of Kupffer cells and NM rinse prevented primary graft non-function in liver grafts from agonal NHBDs.     

MCI-186 (edaravone), a free radical scavenger, attenuates hepatic warm ischemia–reperfusion injury in rats

Hepatic warm ischemia-reperfusion injury (IRI) during hepatectomy and liver transplantation is a major cause of liver dysfunction in which the pathologic role of free radicals is a major concern. To assess the effect of MCI-186 (edaravone) on hepatic IRI, male Wistar rats were subjected to partial hepatic ischemia for 60 min after pretreatment with vehicle (group C) or MCI-186 (group M), or after both MCI-186 pretreatment and additional administration of MCI-186 12 h after reperfusion (group MX). Groups M and MX showed significantly lower levels of serum alanine aminotransferase and hepatic lipid peroxidation than group C, and also significantly lower expression levels of mRNA for cytokines, chemokines and intercellular adhesion molecule-1. There were fewer tissue monocytes and neutrophils in groups M and MX than in group C. These effects were more marked in group MX than in group M. Our findings suggest that treatment with MCI-186 attenuates hepatic IRI in this rat in vivo model.     

依布西林对大鼠无心跳供体肺的保护作用

目的 观察依布西林在大鼠无心跳供体(NHBD)肺保护中的作用.方法 将60只SD大白鼠随机分为A组:有心跳供体(HBD)组;B组:NHBD组;C组:NHBD+依布硒林(Ebselen)组.B组、C组供体处死后维持辅助呼吸,放置室温中30 min,再灌注低钾右旋糖苷(LPD)液.受体鼠行"原位左肺移植术".C组受体在肺移植前1 h给予Ebselen.结果 C组移植后肺顺应性为0.1740±0.0100,结扎右肺门后15、30 min动脉血氧分压分别为(93.97±5.94)、(92.30±6.57)mm-Hg,肺组织丙二醛(MDA)含量为(0.63±0.23)nmol/mg蛋白,肺组织能量代谢物总量为(821.51±29.70)mol/g,与B组比较差异均有统计学意义(P＜0.05).结论 给予受体一定浓度的Ebselen可改善NHBD肺保护作用.

Abstract：

Objective To evaluate the protective effect of ebselen on the rat lungs from non-heartbeating donors (NHBD). Methods Sixty Sprague-Dawley rats were randomly divided into 3 groups:group A, heart-beating donor; group B, NHBD with 30 min of warm ischemia time (WIT); group C, NHBD with 30 min of WIT and administration of ebselen. The donor lungs in groups B and C maintained ventilation at room temperature for 30 min after asystolia and then were flushed with LPD solution. The recipient rats underwent left lung transplantation. The recipients in group C were administered with ebselen 1 h before transplantation. Results All the recipients survived during the observation period. The pulmonary compliance of group C was 0. 1740 ±0. 0100. The PaO2 at 15 min and 30 min after the ligation of the right pulmonary hila was (93.97 ±5.94), (92. 30 ±6. 57) mmHg, respectively. Malondialdehyde (MDA) of the pulmonary tissue was (0. 63 ±0. 23) nmol/mg pro and the energy metabolism was (821.51 ±29.70)mol/g. The difference between group B and group C was significant (P ＜ 0. 05 ). Conclusion The administration of ebselen is a safe and effective treatment in the preservation of the rat lungs from NHBD.     

猪肝移植时无心跳供体耐受热缺血时间的研究

目的 探讨猪肝移植时供肝耐受无心跳热缺血损伤的安全时限。方法 对24头小型家猪根据供肝热缺血时间0、15、30、45min，随机分为4组，而后行原位肝移植，分析移植后肝脏丙氨酸氨基转移酶（ALT）、乳酸脱氢酶（LDH）、三磷酸腺苷（ATP）的含量以及病理变化。结果 WI-45组胆管吻合前无胆汁流出。WI-15、30、45组血清ALT、LDH、肝细胞ATP的含量与WI-0组比较，差异有统计学意义（P〈0．05），WI-15、30组间差异无统计学意义（P〉0．05），但WI-45组与WI-15、30组比较，差异有统计学意义（P〈0．05）。电镜下观察WI-15、30组肝组织呈可复性改变，WI-45组则呈不可逆性改变。结论 猪肝移植时，供肝耐受无心跳热缺血损伤的的安全时限为30min。     

无心跳供肝发生胆道严重缺血性病变的危险因素分析

目的 探讨无心跳供肝发生严重胆道缺血性病变的危险因素.方法 北京朝阳医院2002年7月至2006年6月实施的同种异体原位肝移植病例中排除肝肾联合移植、二次肝移植、供受体ABO血型不符病例,统计人选病例131例,术后随访时间均>180 d.排除混杂因素后采用Logistic回归分析缺血再灌注相关性严重胆道并发症的危险因素.结果 无心跳供肝胆道二次热缺血时间>60 min是术后严重缺血性胆道并发症的独立危险因素.热缺血与冷保存协同作用于供肝,单独或同时延长热缺血、冷保存时间,术后严重缺血性胆道并发症发生率增高.结论 无心跳供肝热缺血或冷保存时间延长的协同作用以及胆道二次热缺血时间>60 min是肝移植术后严重缺血性胆道并发症的危险因素.将热缺血时间带入拟合直线回归方程可预知冷保存时间的相对"安全"时限.     

Ischemic preconditioning prevents free radical production and mitochondrial depolarization in small-for-size rat liver grafts

BACKGROUND: Ischemic preconditioning (IP) renders tissues more tolerant to subsequent longer episodes of ischemia. This study tested whether IP attenuates injury of small-for-size liver grafts by preventing free radical production and mitochondrial dysfunction. METHODS: IP was induced by clamping the portal vein and hepatic artery for 9 min. Livers were harvested 5 min after releasing the clamp. Mitochondrial polarization and cell death were assessed by intravital confocal/multiphoton microscopy of rhodamine 123 (Rh123) and propidium iodide. Free radicals were trapped with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone and measured using electron spin resonance. RESULTS: After quarter-size liver transplantation, alanine aminotransferase, serum bilirubin, necrosis, and apoptosis all increased. IP blocked these increases by more than 58%. 5-Bromo-2'-deoxyuridine labeling and increases of graft weight were only approximately 3% and 0.2% in quarter-size grafts without IP, respectively, but increased to 32% and 60% in ischemic-preconditioned grafts, indicating better liver regeneration. Eighteen hours after implantation, viable cells with depolarized mitochondria in quarter-size grafts were 15 per high power field, and dead cells were less than 1 per high power field, indicating that depolarization preceded necrosis. A free radical adduct signal was detected in bile from quarter-size grafts. IP decreased this free radical formation and prevented mitochondrial depolarization. IP did not increase heat shock proteins 10, 27, 32, 60, 70, 72, 75 and Cu/Zn-superoxide dismutase (SOD) but increased heat shock protein-90, a chaperone that facilitates protein import into mitochondria, and mitochondrial Mn-SOD. CONCLUSION: Taken together, IP decreases injury and improves regeneration of small-for-size liver grafts, possibly by increasing mitochondrial Mn-SOD, thus protecting against free radical production and mitochondrial dysfunction.