UW保存液中不同缺血时间对肝移植效果的影响

自1963年3月1日Thomas Starzl施行了人类历史上第一例肝移植至今,肝移植已有40多年的历史;作为治疗终末期肝脏疾病的唯一有效手段,其效果受到许多因素的影响,缺血时间即是其中之一。     

肝脏热,冷缺血和再灌注损伤及其机制

由于环孢霉素运用,手术技术改进,肝移植成为治疗先天性或后天性晚期肝病唯一有效的手段。目前全世界共施行人体原位肝移植4000余例,但由于缺乏有效监测手段,供肝移植后原发性无功能(Primary Non-Function,PNF)发生率达23％,患者若未能及时行再移植手术或其他积极有效治疗,其死亡率可高达     

乙肝核心抗体阳性供肝在肝移植中的应用

背景：现已经证实使用anti-HBc（＋）供肝会使移植后乙肝复发的风险,但anti-HBc（＋）供肝的应用明显缓解了供肝的相对匮乏。目的：分析应用anti-HBc（＋）供肝移植后乙肝复发风险及有效的预防措施。方法：应用计算机检PubMed数据库中1994-01/2009-12关于anti-HBc（＋）供肝文章,在标题和摘要中以＂Hepatitis B core antibody;donor;liver transplantation＂为检索词进行检索。选择与anti-HBc供肝相关文章。初检得到109篇文献,根据纳入标准选择48篇文章进行综述。结果与结论：HBsAg（＋）患者接受anti-HBc（＋）供肝移植术后乙肝复发率为11%,生存率为67%～100%,与HBsAg（＋）受者接受anti-HBc（-）供肝相似。HBsAg（-）受者接受anti-HBc（＋）供肝总体感染率为19%,其中未感染过乙肝受者移植术后乙肝感染率为48%,感染过乙肝受者后感染率为15%。未感染乙肝与感染过乙肝受者移植后采取有效预防措施后感染率分别为3%,12%。采用HBIG、拉米夫定、联合用药的移植后感染率分别为19%,2.6%,2.8%。提示,采用anti-HBc（＋）供肝做为供体是安全的,尤其是用在HBsAg（＋）、anti-HBc（＋）、anti-HBs（＋）受者。而HBsAg（-）受者移植后接受拉米夫定可以有效复发乙肝感染。     

不同热缺血时间下供心冷保存期间心肌ATP含量变化

目的 探讨不同热缺血时间下供心冷保存期间ＡＴＰ的下降程度,以确定不同热因时间下供心冷保存的时限。方法 新西兰在白兔１８只,随机分成１,２,３组（ｎ＝６）,开胸后,取左室前壁心肌测ＡＴＰ,按常规取心方法分别在阻断主动脉后（热缺血）０ｍｉｎ（１组）,５ｍｉｎ（２组）,１０ｍｉｎ（３组）取下心脏,保存于４℃的Ｒｉｎｇｅｒ’ｓ液中,３０ｍｉｎ,１ｈ,２ｈ,３ｈ,４ｈ取左室心肌测ＡＴＰ（其中１组加测５ｈ）。     

冷保存方法构建的大鼠肝移植模型

背景:肝移植是终末期肝病最为有效的治疗手段,移植后胆病是制约肝移植发展的主要障碍之一.目的:建立稳定的大鼠冷保存肝移植模型,探讨冷保存对肝移植后肝脏胆管的影响.方法:120只雄性SD大鼠随机分为4组,按照组内随机配对的原则,体质量相对较轻的大鼠做为供体,供肝置于4℃ UW液中分别保存2,8,16 h后行原位肝移植.在两套袖法基础上,以支架法建立动脉化大鼠原位肝移植模型,供受体肝总动脉采用改良支架法进行端端吻合,重建肝动脉血供.记录移植手术时间及移植成功率,并分别于移植后3,7 d检测血清转氨酶、总胆红素、碱性磷酸酶水平,同时观察组织病理学改变.结果与结论:实验共完成55例次大鼠原位肝移植手术,手术成功率为93%.冷保存2,8,16 h组术后7 d存活率分别为100%(9/9),83%(10/12),73%(8/11).随着冷保存时间的延长,肝功能及肝内胆管损伤加重,胆管组织病理学评分显示各组间差异有显著性意义(P<0.05).其中供肝冷保存16 h大鼠肝移植模型既有较高的手术存活率又有严重的胆管损伤,是研究冷保存对肝移植胆管病影响的较好模型.     

胎肝移植的供肝采集,修剪和保存

通过5例胎龄为7月的胚胎所进行的实验,用电镜和酶组织化学观察,结果证明我们设计的一套胎肝灌注、切取、修剪、保存方法是可行的。用4℃平衡液加改良SacksⅡ液经脐静脉和髂总动脉插管进行原位冷灌注多脏器联合切取后,在0～4℃条件下修剪保存胎肝的方法可保存胎肝活力9小时。     

大鼠肝移植不同供肝切取技术对供肝质量的影响

目的:探讨在大鼠肝移植供肝切取过程中不同方法对供肝质量的影响。方法:SD大鼠120只,随机分成3组,即标准法组(A组)、快速法组(B组)和改良快速法组(C组),每组各行20对大鼠原位肝移植,观察术中资料和术后3周存活率及冷灌洗后肝脏形态学改变。结果:在供肝切取手术时间上C组明显较A、B两组为短(P<0.05),其术后3周存活率也高于A组(P<0.05)。光镜下C组与A、B两组相比肝窦内未见红细胞。结论:标准法和快速法适合于刚建立大鼠肝移植模型的单位,为提高供肝质量、改善术后存活率应选择改良快速法。     

乙肝核心抗体阳性供肝在肝移植中的应用

背景:现已经证实使用anti-HBc(+)供肝会使移植后乙肝复发的风险,但anti-HBc(+)供肝的应用明显缓解了供肝的相对匮乏.目的:分析应用anti-HBc(+)供肝移植后乙肝复发风险及有效的预防措施.方法:应用计算机检PubMed 数据库中1994-01/2009-12 关于anti-HBc(+)供肝文章,在标题和摘要中以"Hepatitis B coreantibody; donor;liver transplantation"为检索词进行检索.选择与anti-HBc 供肝相关文章.初检得到109 篇文献,根据纳入标准选择48 篇文章进行综述.结果与结论:HBsAg(+)患者接受anti-HBc(+)供肝移植术后乙肝复发率为11%,生存率为67%～100%,与HBsAg(+)受者接受anti-HBc(-)供肝相似.HBsAg(-)受者接受anti-HBc(+)供肝总体感染率为19%,其中未感染过乙肝受者移植术后乙肝感染率为48%,感染过乙肝受者后感染率为15%.未感染乙肝与感染过乙肝受者移植后采取有效预防措施后感染率分别为3%,12%.采用HBIG、拉米夫定、联合用药的移植后感染率分别为19%,2.6%,2.8%.提示,采用anti-HBc(+)供肝做为供体是安全的,尤其是用在HBsAg(+)、anti-HBc(+)、anti-HBs(+)受者.而HBsAg(-)受者移植后接受拉米夫定可以有效复发乙肝感染.     

Plasma thrombopoietin level after liver transplantation: relationship to cold ischemia time and coagulation parameters

Objective: To determine the relation between thrombopoietin (Tpo) levels following orthotopic liver transplantation (OLT), cold ischemia time and postoperative peripheral blood platelet count and prothrombin activity.¶Design: Prospective clinical study.¶Setting: Intensive care unit.¶Patients: Fourteen patients with uncomplicated postoperative course after OLT.¶Measurements and results: Plasma Tpo, as quantified by enzyme immunoassay, rose significantly from 194.9 ± 45.7 pg/ml on day 1 after OLT to a peak value of 500.7 ± 94.1 pg/ml on day 5 while platelet count was below normal values. Then the platelet count increased and reached normal values while Tpo decreased to normal. The rise of Tpo levels was associated with normalization of prothrombin time but peak Tpo concentrations were in inverse correlation with cold ischemia times.¶Conclusion: The extent of production of Tpo in the liver graft following OLT is affected by cold ischemia time. This observation may be applicable in the prevention of bleeding complications associated with postoperative thrombocytopenia.     

无心跳供肾模型大鼠的安全热缺血时限

背景：近年来心脏停博供体已然成为最有潜力的移植供体来源。目前有关于供肾安全热缺血期的时限研究各报道差异较大,更未上升到安全时限的理论高度。目的：探讨建立大鼠无心跳供肾模型的实验方法,并分析此供体耐受热缺血的安全时限。方法：采用断延髓法建立大鼠无心跳供肾模型,并按肾移植供体经历的热缺血时间0,10,30,60min将其分为4组。观察离体肾脏经过不同热缺血期后,供肾的组织病理学及肾组织丙二醛含量变化。结果与结论：光镜下观察,热缺血30min以前,肾脏病理改变为可逆性改变,随着热缺血时间的延长病理改变向着不可逆方向发展。与无热缺血期比较,热缺血10min组、30min组及60min组的丙二醛含量均有所升高（P〈0.05）;与热缺血10min组比较,热缺血30min组及60min组丙二醛含量明显升高（P〈0.05）;但热缺血30min与60min组差异无显著性意义（P〉0.05）。提示可成功采用断延髓法建立无心跳供肾模型,并在此基础上,结合供肾的病理结果和肾组织丙二醛含量变化,综合评定无心跳供肾所能耐受的安全热缺血时限为30min。     

无心跳供肾模型大鼠的安全热缺血时限

背景:近年来心脏停博供体已然成为最有潜力的移植供体来源.目前有关于供肾安全热缺血期的时限研究各报道差异较大,更未上升到安全时限的理论高度.目的:探讨建立大鼠无心跳供肾模型的实验方法,并分析此供体耐受热缺血的安全时限.方法:采用断延髓法建立大鼠无心跳供肾模型,并按肾移植供体经历的热缺血时间0,10,30,60 min将其分为4组.观察离体肾脏经过不同热缺血期后,供肾的组织病理学及肾组织丙二醛含量变化.结果与结论:光镜下观察,热缺血30 min以前,肾脏病理改变为可逆性改变,随着热缺血时间的延长病理改变向着不可逆方向发展.与无热缺血期比较,热缺血10 min组、30 min组及60 min组的丙二醛含量均有所升高(P < 0.05);与热缺血10 min组比较,热缺血30 min组及60 min组丙二醛含量明显升高(P < 0.05);但热缺血30 min与60 min组差异无显著性意义(P > 0.05).提示可成功采用断延髓法建立无心跳供肾模型,并在此基础上,结合供肾的病理结果和肾组织丙二醛含量变化,综合评定无心跳供肾所能耐受的安全热缺血时限为30 min.     

Intracranial bypass grafts for vertebral-basilar ischemia

Fourteen operations in which an occipital branch of the external carotid artery was anastomosed to the posterior inferior cerebellar artery were performed for occlusions or inaccessible stenotic lesions of the vertebral arteries proximal to the site of origin of the posterior inferior cerebellar artery. Eight patients (group 1) had no major focal reurologic deficit but were considered to be at high risk for a posterior circulation infarct; six patients (group 2) had been severely or moderately disabled before the operation. Postoperative angiography revealed that 13 of the 14 grafts were patent. In 9 of the 13 patent grafts, the bypass graft served as the sole or major blood supply of the vertebral-basilar system; in 4, flow was limited to the distribution of the posterior inferior cerebellar artery. Five of the eight patients in group 1 have returned to full employment or normal retired life. Two of the six patients in group 2 have resumed normal activities with only minimal neurologic dysfunction. This procedure may have a role in the management of highly selected patients suffering from vertebral-basilar ischemia, and it may be useful in the management of selected aneurysms in the vertebral-basilar system.     

睾丸移植冷缺血时间与睾丸缺血再灌注损伤的关系

背景:睾丸移植是短时间热缺血损伤后快速进入冷缺血的过程,冷缺血时间的长短对睾丸生精功能是否存在影响目前还缺乏明确证据.目的:模拟临床睾丸移植缺血再灌注兔模型,探索睾丸移植冷缺血时间与睾丸缺血再灌注损伤的相关性.设计、时间及地点:分组设计,对比观察,于2006-05/12在武汉大学人民医院动物实验中心完成.材料:雄性3～6月龄大白兔,体质量2.5～3.5 kg,方法:采用自行设计的模拟临床睾丸移植过程的冷缺血灌注模型,对20只大白兔芹侧的睾丸采用0～4℃高渗枸橼酸腺嘌呤液恒压灌注,并浸泡于0～4℃生理盐水中低温保存,于冷缺血后1,2,4,6 h开放左侧睾丸血流.术后24 h取双侧睾丸,以右侧睾丸作自身对照.主要观察指标;苏木精-伊红染色及Johnsen评分判断睾丸损伤程度.丙二醛检测试剂盒检测每克睾丸组织中丙二醛含量.TUNEL法检测睾丸凋亡指数.结果:冷缺血1 h后可见睾丸生精上皮结构稍紊乱,2 h后生精上皮开始脱落,管腔内精子数量减少,4 h后生精上皮明显变薄,仅见少量的精子细胞,6 h后生精上皮仅可见少量精母细胞,部分呈唯Sertoli细胞表现.随冷缺血时间的延长,睾丸缺血再灌注损伤逐渐加重,Johnsen评分分值逐渐下降.睾丸组织冷缺血2 h后再灌注24 h的丙二醛水平明显高于自身对照组(P<0.05),至4 h达到最高.凋亡细胞数量随冷缺血时间的延长逐渐增多,凋亡指数高于自身对照(P<0.05).结论:冷缺血后4h再灌注会造成睾丸生精上皮的严重损害,提示睾丸移植前冷缺血时间应当控制在4 h以内.     

Controlled oxygenated rewarming as novel end‐ischemic therapy for cold stored liver grafts. A randomized controlled trial

Abrupt return to normothermia has been shown a genuine factor contributing to graft dysfunction after transplantation. This study tested the concept to mitigate reperfusion injury of liver grafts by gentle warming‐up using ex vivo machine perfusion prior to reperfusion. In a single center randomized controlled study, livers were assigned to conventional static cold storage (SCS) alone or to SCS followed by 90 min of ex vivo machine perfusion including controlled oxygenated rewarming (COR) by gentle and protracted elevation of the perfusate temperature from 10°C to 20°C. Primary outcome mean peak aspartate aminotransferase (AST) was 1371 U/L (SD 2871) after SCS versus 767 U/L (SD 1157) after COR (p = 0.273). Liver function test (LiMAx) on postoperative day 1 yielded 187 μg/kg/h (SD 121) after SCS, but rose to 294 μg/kg/h (SD 106) after COR (p = 0.006). Likewise, hepatic synthesis of coagulation factor V was significantly accelerated in the COR group immediately after transplantation (103% [SD 34] vs. 66% [SD 26]; p = 0.001). Fewer severe complications (Clavien‐Dindo grade ≥3b) were reported in the COR group (8) than in the SCS group (15). Rewarming/reperfusion injury of liver grafts can be safely and effectively mitigated by controlling of the rewarming kinetics prior to blood reperfusion using end‐ischemic ex vivo machine perfusion after cold storage.

Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Experimentally, evidence suggests that abrupt rewarming upon reperfusion after cold storage compromises graft recovery. This “rewarming injury” could be significantly alleviated by modifying the slope of temperature increase in the organ graft upon isolated machine perfusion. This controlled oxygenated rewarming gently restitutes metabolic turnover rates and prevents the abrupt reactivation of a not yet equilibrated cellular metabolism. WHAT QUESTION DID THIS STUDY ADDRESS? This study is the first randomized controlled trial to address the question if and to which end the method of controlled oxygenated rewarming can be translated into clinical liver transplantation. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? This study shows that controlled oxygenated rewarming during ex vivo machine perfusion can be regarded as a safe and easily implementable novel tool in liver preservation, likely to improve early functional outcome after transplantation. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? The addition of a brief machine perfusion protocol with a controlled rise in temperature is an easily implementable method in clinical routine and may contribute to improved organ recovery after transplantation.     

Sustained tolerance to lipopolysaccharide after liver ischemia-reperfusion injury

Liver ischemia-reperfusion injury (IR) would be expected to alter the capacity of previously ischemic as well as continuously perfused segments that are exposed to circulating inflammatory mediators to respond to a subsequent infectious insult. IR is reported to induce tolerance to subsequent endotoxin stimulation if the lipopolysaccharide (LPS) challenge is delayed until the late, neutrophil-mediated phase of reperfusion. Whether ischemic or perfused liver is differentially affected and whether LPS-tolerance may be overcome by increasing exposure is unknown. We hypothesized that late tolerance after IR reflects a refractory state in which the liver's expression of pro-inflammatory mediators in response to secondary LPS is limited. Precision-cut tissue culture methodology was used to investigate the capacity of rabbit liver to respond to a spectrum of LPS stimulation 24 h after partial IR. Slices from normal liver showed a dose-dependent response to LPS for tumor necrosis factor (TNF-alpha) expression. Slices from both previously ischemic and continuously perfused lobes retained dose responsiveness for TNF-alpha, although TNF-alpha was significantly decreased at high LPS concentrations compared with normal liver. Ischemic liver sustained this blunted response despite extended exposure to LPS, whereas perfused slices recovered responsiveness to high dose LPS with prolonged stimulation. IR induced interleukin-8 in both ischemic and perfused liver, but secondary LPS stimulation did not augment interleukin-8 expression. Hepatic IR induces a late tolerance to secondary LPS challenge in locally ischemic tissue that cannot be overcome by increasing LPS exposure. Nonischemic liver exposed to the systemic effects of IR injury, however, retains a capacity to respond to LPS with sufficient stimulation.     

Effects of endotoxin tolerance on liver function after hepatic ischemia/reperfusion injury in the rat

OBJECTIVE: It is known that endotoxin tolerance prevents lethality after ischemia/reperfusion injuries (e.g., myocardial infarction) in laboratory animals. We used a rat model of partial hepatic ischemia/reperfusion to investigate whether endotoxin tolerance prevents associated lethality and disorders of liver function. DESIGN: Prospective animal study. SETTING: University research facility. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Hepatic ischemia was initiated by atraumatic clipping across the portal venous and hepatic arterial blood supply to the left lateral lobe for 90 mins. The common bile duct was canalized, and in a second set of experiments the bile duct of the left lateral lobe was canalized selectively. Bile flow, bile acids, and transaminases were determined during ischemia and 300 mins of reperfusion in endotoxin-tolerant and -nontolerant rats. MEASUREMENTS AND MAIN RESULTS: Endotoxin-nontolerant animals showed a 50% lethality after hepatic ischemia/reperfusion injuries. All endotoxin-tolerant rats survived and did not react with any change in bile flow, showing a constant flow. The amount of bile acids in the common bile duct was reduced during ischemia and regained the concentrations of sham-operated animals 60 mins after reperfusion. From 180 mins after reperfusion, the difference between endotoxin-tolerant and -nontolerant animals was statistically significant. When bile acid concentration was determined in the ischemic left lateral lobe, ischemia/reperfusion was found to significantly decrease in endotoxin-nontolerant rats 60 mins after reperfusion. In contrast, endotoxin-tolerant rats produced normal amounts of bile acids 60 mins after reperfusion. At 120 mins after reperfusion, the amount of bile acids in the formerly ischemic left lateral lobe was more than normal. CONCLUSIONS: In this model of partial hepatic ischemia/reperfusion, endotoxin tolerance prevents ischemia/reperfusion injury-associated lethality and local disorders of liver function. This phenomenon induced by endotoxin tolerance may be useful in liver surgery to prevent ischemia/reperfusion injury.