Long-term normothermic machine perfusion of fatty livers:towards transplanting untransplantable livers?

Since the very early days of clinical liver transplantation(LT),transplant surgeons and professionals have been confronted with the increased risk of failure associated with the use of fatty liver grafts(1).Notwithstanding the wide variability in steatosis assessment across different centers and pathologists(2),utilization of livers with moderate(≥30%)or severe(≥60%)macrovesicular steatosis has been consistently associated with an increased risk of primary non-function,early allograft dysfunction,acute kidney injury,as well as inferior graft and patient survival(3).The mechanisms behind the increased susceptibility of steatotic livers to ischemia-reperfusion injury(IRI)are multiple,including disturbances to microcirculation due to sinusoidal narrowing,increased oxidative stress upon reperfusion and enhanced lipid peroxidation,leading to an increased release of inflammatory mediators like IL6,IL1βand so-called damage associated molecular patterns(DAMP),like cell-free DNA and mitochondrial DNA.Histologically,this is reflected by hepatocyte death by necrosis rather than apoptosis,pseudopeliotic steatosis(i.e.,the expulsion of lipid droplets into the extracellular space)and lately,by tissue remodelling and fibrosis(4,5).

Mechanisms of autophagy activation in endothelial cell and their targeting during normothermic machine liver perfusion

Ischaemia-reperfusion injury(IRI) is the leading cause of injury seen in the liver following transplantation. IRI also causes injury following liver surgery and haemodynamic shock. The first cells within the liver to be injured by IRI are the liver sinusoidal endothelial cells(LSEC). Recent evidence suggests that LSEC coordinate and regulates the livers response to a variety of injuries. It is becoming increasingly apparent that the cyto-protective cellular process of autophagy is a key regulator of IRI. In particular LSEC autophagy may be an essential gatekeeper to the development of IRI. The recent availability of liver perfusion devices has allowed for the therapeutic targeting of autophagy to reduce IRI. In particular normothermic machine liver perfusion(NMP-L) allow the delivery of pharmacological agents to donor livers whilst maintaining physiological temperature and hepatic flow rates. In this review we summarise the current understanding of endothelial autophagy and how this may be manipulated during NMP-L to reduce liver IRI.

Liver graft preservation methods during cold ischemia phase and normothermic machine perfusion

The growing demand for donor organs requires measures to expand donor pool.Those include extended criteria donors, such as elderly people, steatotic livers,donation after cardiac death, etc. Static cold storage to reduce metabolic requirements developed by Collins in late 1960 s is the mainstay and the golden standard for donated organ protection. Hypothermic machine perfusion provides dynamic organ preservation at 4°C with protracted infusion of metabolic substrates to the graft during the ex vivo period. It has been used instead of static cold storage or after it as short perfusion in transplant center. Normothermic machine perfusion(NMP) delivers oxygen, and nutrition at physiological temperature mimicking regular environment in order to support cellular function. This would minimize effects of ischemia/reperfusion injury.Potentially, NMP may help to estimate graft functionality before implantation into a recipient. Clinical studies demonstrated at least its non-inferiority or better outcomes vs static cold storage. Regular grafts donated after brain death could be safely preserved with convenient static cold storage. Except for prolonged ischemia time where hypothermic machine perfusion started in transplant center could be estimated to provide possible positive reconditioning effect. Use of hypothermic machine perfusion in regular donation instead of static cold storage or in extended criteria donors requires further investigation. Multicenter randomized clinical trial supposed to be completed in December 2021. Extended criteria donors need additional measures for graft storage and assessment until its implantation. NMP is actively evaluating promising method for this purpose.Future studies are necessary for precise estimation and confirmation to issue clinical practice recommendations.

Routine utilization of machine perfusion in liver transplantation:Ready for prime time?

The last decade has been notable for increasing high-quality research and dramatic improvement in outcomes with dynamic liver preservation.Robust evidence from numerous randomized controlled trials has been pooled by meta-analyses,providing the highest available evidence on the protective effect of machine perfusion(MP)over static cold storage in liver transplantation(LT).Based on a protective effect with less complications and improved graft survival,the field has seen a paradigm shift in organ preservation.This editorial focuses on the role of MP in LT and how it could become the new“gold standard”.Strong collaborative efforts are needed to explore its effects on long-term outcomes.

Machine perfusion and the prevention of ischemic type biliary lesions following liver transplant:What is the evidence?

The widespread uptake of different machine perfusion(MP)strategies for liver transplant has been driven by an effort to minimize graft injury.Damage to the cholangiocytes during the liver donation,preservation,or early posttransplant period may result in stricturing of the biliary tree and inadequate biliary drainage.This problem continues to trouble clinicians,and may have catastrophic consequences for the graft and patient.Ischemic injury,as a result of compromised hepatic artery flow,is a well-known cause of biliary strictures,sepsis,and graft failure.However,very similar lesions can appear with a patent hepatic artery and these are known as ischemic type biliary lesions(ITBL)that are attributed to microcirculatory dysfunction rather than main hepatic arterial compromise.Both the warm and cold ischemic period duration appear to influence the onset of ITBL.All of the commonly used MP techniques deliver oxygen to the graft cells,and therefore may minimize the cholangiocyte injury and subsequently reduce the incidence of ITBL.As clinical experience and published evidence grows for these modalities,the impact they have on ITBL rates is important to consider.In this review,the evidence for the three commonly used MP strategies(abdominal normothermic regional perfusion[A-NRP],hypothermic oxygenated perfusion[HOPE],and normothermic machine perfusion[NMP])for ITBL prevention has been critically reviewed.Inconsistencies with ITBL definitions used in trials,coupled with variations in techniques of MP,make interpretation challenging.Overall,the evidence suggests that both HOPE and A-NRP prevent ITBL in donated after circulatory death grafts compared to cold storage.The evidence for ITBL prevention in donor after brain death grafts with any MP technique is weak.

Comprehensive Approach to Assessment of Liver Viability During Normothermic Machine Perfusion

Liver transplantation is the most effective treatment of advanced liver disease,and the use of extended criteria donor organs has broadened the source of available livers.Although normothermic machine perfusion(NMP)has become a useful tool in liver transplantation,there are no consistent criteria that can be used to evaluate the viability of livers during NMP.This review summarizes the criteria,indicators,and methods used to evaluate liver viability during NMP.The shape,appearance,and hemodynamics of the liver can be analyzed at a macroscopic level,while markers of liver injury,indicators of liver and bile duct function,and other relevant indicators can be evaluated by biochemical analysis.The liver can also be assessed by tissue biopsy at the microscopic level.Novel methods for assessment of liver viability are introduced.The limitations of evaluating liver viability during NMP are discussed and suggestions for future clinical practice are provided.

Standardization is needed in reporting risk and outcomes of machine perfusion in liver transplantation

We congratulate Yamamoto et al.on their study“Impact of Portable Normothermic Machine Perfusion for Liver Transplantation from Adult Deceased Donors”(1).The surge of interest in normothermic machine perfusion(NMP)in the United States(US)has generated considerable enthusiasm.The authors present generally exciting results from their single-center analysis of 541 liver transplantations.Among these,469 were from donors after brain dead(DBD);58(12.4%)received NMP and 411 static cold storage(SCS,87.6%).Seventy-two transplants were from donors after circulatory death(DCD);52(72.2%)received NMP(device-to-donor)vs.20 SCS(27.8%).

Does ischemia-free machine perfusion lead to early allograft dysfunction (EAD) free liver transplantation?

Normothermic machine perfusion(NMP)is an innovative technique used in solid organ transplantation that involves perfusing the organ with specialized solution or leukocyte depleted red blood cells at near-normal body temperature aiming at mimicking physiological conditions,and providing optimal conditions for organ preservation which leads to reduced risk of ischemia reperfusion injury(IRI)when compared to the standard static cold storage(SCS)(1).One of the key advantages of NMP is its ability to assess the quality of the organ in real-time and assessment of the organ performance prior to transplantation.By continuously monitoring parameters such as blood flow,oxygen consumption,and lactate production,clinicians can evaluate the viability of the organ and make more informed decisions about its suitability for transplantation.This real-time assessment can help reduce the risk of transplanting organs that may not function optimally or have a higher likelihood of complications post-transplant.Additionally,NMP may expand the pool of donor organs by allowing for the use of organs that may have been deemed marginal or unsuitable for transplantation using traditional methods(1,2).

The conclusion of reducing acute rejection after liver transplantation by machine perfusion should be extrapolated with caution

There is a theory that the unavoidable graft damage caused by ischemia-reperfusion injury(IRI)during liver transplantation(LT)can lead to severe IRI-related inflammation and trigger an early activation of the innate immune response mediated by T-cells,which potentially worsening the acute cellular rejection(ACR)cascade.As a result,machine perfusion(MP)has been placed great expectations for the potential to diminish post-LT ACR and other related immune responses by alleviating IRI through removing harmful substances and restoring cellular metabolism homeostasis(1,2).However,there has been much debate about MP’s benefits on ACR as relative data is limited.

肾脏离体机械灌注治疗新进展

肾移植仍然是终末期肾病病人的金标准治疗方法。为了满足不断增长的器官需求,越来越多地使用心脏死亡捐献者(DCD)或扩大标准捐赠者(ECD)的器官。这些边缘器官发生严重缺血再灌注损伤的风险高,移植后原发性无功能、移植物功能延迟恢复和远期移植物存活率降低的发生率增加。肾脏离体机械灌注(EVMP)与传统静态冷保存相比,可以更好地保护供肾。EVMP还可以作为动态应用药物或基因治疗的平台,以进一步改善肾脏移植疗效。本综述概述了目前EVMP治疗的新策略,包括间充质干细胞疗法、基因疗法、纳米技术、抗感染剂、气体治疗、溶栓药、血型转化等。

常温机械灌注技术在离断肢体保存中的作用研究进展

肢体离断伤在临床工作中较为常见,对离断肢体实施安全有效的保护是肢体再植成功的关键。常温机械灌注技术已经在器官移植领域取得很大突破,可长时间维持器官和组织的活性功能,延长其保存时间,在大动物模型以及临床应用中得到了很好的验证。同时这项技术有望为离断肢体的保存和功能恢复提供新的参考。因此,本文就静态冷保存在离断肢体保存中存在的问题、机械灌注的发展历程、离断肢体常温机械灌注的临床应用现状以及有待解决问题进行综述,并展望其发展方向和临床应用前景,以期推动该技术在临床广泛应用。

人源化基因修饰猪血红细胞常温机械灌注技术对人断肢的保护作用

目的探讨人源化基因修饰猪血红细胞常温机械灌注对人离断肢体的保存作用。方法采用人源化基因修饰猪血红细胞灌注人离断肢体6 h。每小时取灌注液检测血氧分压、Na^(+)、K^(+)、Ca^(2+)、pH值、葡萄糖、乳酸及肌酸激酶水平,取浅层屈肌检测肿瘤坏死因子(TNF)-α、白细胞介素(IL)-2、IL-1变化。灌注0 h、6 h取适量前臂浅层屈肌,进行病理学检查,观察骨骼肌细胞间隙及糖原消耗情况。每2 h取适量前臂血管,检测血管内皮细胞凋亡情况。灌注开始前及灌注6 h行X线造影,观察指端末梢血管充盈度。结果灌注过程中氧分压处于正常范围。Na^(+)在1 h出现高峰,达到138.7 mmol/L,之后波动于正常范围;K^(+)在2 h出现高峰,为6.08 mmol/L,之后下降,波动于正常范围;Ca^(2+)在4 h达高峰,为1.03 mmol/L。葡萄糖在灌注开始时逐渐降低,灌注2 h时达最低值17.7 mmol/L,之后维持动态平衡。pH值在灌注6 h降低为7.28,乳酸水平在灌注1 h时升高,达到9.6 mmol/L,之后逐渐呈下降趋势。肌酸激酶水平从灌注开始升高,于2 h到达高峰,为20030 U/L,之后下降,在灌注结束时保持稳定。灌注结束肌纤维形态正常,肌纤维间隙略有增大,骨骼肌糖原未见明显蓄积。灌注0 h时血管内皮凋亡细胞数量较多,6 h时凋亡细胞数量减少。灌注0 h时血管充盈明显,6 h时指间部分血管充盈程度减低。结论人源化基因修饰猪血红细胞常温机械灌注人断肢能持续稳定的供能供氧,保持灌注液离子酸碱度平衡,维持细胞正常代谢,对离断肢体有一定保护作用。

新型常温机械灌注装置保存猪DCD供肾的实验研究

目的构建新型常温机械灌注(normothermic machine perfusion,NMP)系统,利用猪自体肾移植模型,探讨NMP改善猪心脏死亡器官捐献(donation after cardiac death,DCD)供肾保存质量及相关机制。方法选用健康巴马小型猪10只,随机分为两组,在原位夹闭右肾动脉30分钟后,UW液低温灌注,A组(n=5)以NMP保存8小时,B组(n=5)以UW液4℃低温保存8小时,然后两组均行自体肾移植术,比较术后血清生化指标、肾组织病理变化、细胞因子的表达情况等指标。结果两组均成功在经过30分钟热缺血和8小时保存后,进行自体肾移植。血流开放1小时后,A组肾组织免疫组化肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、天冬氨酸特异性半胱氨酸蛋白酶-3(cysteinyl aspartate specific proteinase-3,Caspase-3)、内毒素(endotoxin-1,ET-1)水平明显低于B组,TUNEL法测定凋亡细胞比例也明显低于B组,两组之间差异有统计学意义(P<0.05);血流开放6小时后,A组血清肌酐(serum creatinine,SCr)、TNF-α、Caspase-3水平明显低于B组,两组之间差异有统计学意义(P<0.05);开放后肾脏活检常规病理检查提示,A组肾脏损伤程度明显减轻。结论 NMP保存可明显减轻猪DCD供肾的缺血/再灌注损伤;其保护机制与NMP可模拟器官的生理环境,维持充足能量与氧气供应,减少保存期间和再灌注后炎性细胞因子释放等有关。

肝脏常温机械灌注修复及评估系统的研制

介绍了一种肝脏常温机械灌注修复及评估系统。系统由肝脏常温氧合机械灌注装置、荧光成像系统及组织测氧仪组成。常温氧合机械灌注装置可对供肝进行持续灌注并实时监测调控灌注参数,荧光成像系统通过识别肝脏代谢的吲哚菁绿来评估供肝微循环情况及肝细胞代谢功能,组织测氧仪可实时监测肝组织氧分压变化评估细胞耗氧状态。

常温机械灌注对大鼠心脏死亡器官捐献供肝微循环保护作用的研究

目的探讨常温机械灌注(normothermic machine perfusion,NMP)对大鼠心脏死亡器官捐献(donation after circulatory death,DCD)供肝微循环发挥的作用。方法采用夹闭SD大鼠胸主动脉热缺血30 min获取DCD供肝;在体外建立大鼠NMP系统。根据不同的保存供肝方式,将实验分为:正常(Normal)组(n=6),留取血清及肝脏待用;NMP组(n=30),保存4、6和8 h后收集肝脏标本,灌注后0、2、4、6 h和8 h收集流入道、流出道灌注液待检;静态冷保存(SCS)组(n=6),肝脏以20 ml 4℃UW液冲出肝内血液,并于UW液中4℃SCS 6 h后收集肝脏标本。采用生物化学方法检测流出道灌注液的肝功能;HE染色观察肝组织病理学改变;透射电镜观察肝细胞超微结构;TUNEL检测肝脏细胞凋亡情况;Western blot检测肝脏组织内皮素-1(endothelin-1,ET-1)、内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、血管性血友病因子(von Willebrand factor,vWF)、细胞间黏附分子(intercellular adhesion molecule-1,ICAM-1)和血管间黏附分子-1(intervascular adhesion molecule-1,VCAM-1)的表达情况。结果与SCS比较,NMP能显著改善DCD供肝组织学损伤,Suzuki评分NMP组(3.40±0.55)显著低于SCS组(7.00±0.71,F=229.75,P<0.05);减轻肝细胞凋亡,NMP组凋亡细胞数(9.80±1.48)显著低于SCS组(33.40±4.39,F=166.58,P<0.05);同时能修复肝细胞线粒体损伤,NMP组不可逆损伤线粒体数量(1.60±0.55)显著低于SCS组(2.80±0.45,F=36.29,P<0.05)。进一步发现,NMP可改善DCD肝脏微循环:①抑制细胞间黏附,改善内皮细胞损伤,与SCS比较,NMP显著抑制肝内ICAM-1(F=1728.45,P<0.05)、VCAM-1(F=254.72,P<0.05)和vWF(F=595.30,P<0.05)的表达;②改善肝脏ET-1/NOS平衡和微循环灌注,与SCS相比,NMP显著抑制肝内ET-1(F=1372.51,P<0.05)、iNOS(F=1102.20,P<0.05)的表达,促进eNOS(F=271.66,P<0.05)的表达。结论NMP能够改善大鼠DCD供肝质量,其机制可能通过抑制细胞间黏附,改善窦内皮损伤及微循环灌注发挥保护作用的。

基于常温机械灌注不中断血流保存供肝肝移植的手术配合

目的总结基于常温机械灌注不中断血流保存供肝的肝移植手术配合要点。方法 10例患者在常温机械灌注保存供肝下进行肝移植手术,做好供肝获取和保护,不间断加强血流供肝获取的手术配合,准备灌注液、灌注机器,做好常温机械灌注供肝期的安全管理,手术期体温的控制以及供肝植入手术的护理。结果 10例手术均顺利完成,术后恢复良好,出院前肝功能各项指标均恢复正常水平,未发生手术并发症,术后每个月随访复查肝功能,10例患者均未发生急性排斥反应,有1例患者术后1个月复查丙氨酸氨基转移酶(ALT)偏高,予护肝等综合治疗后恢复正常。结论常温机械灌注保存供肝在肝移植手术应用效果显著,充分的术前准备,医护人员扎实的专业技术及密切的合作,是提高手术效率及保证手术顺利进行的关键。

心脏移植的发展现状和新挑战

心脏移植是终末期心力衰竭患者的首选治疗。供者不足一直以来都是限制心脏移植数量增长的主要问题,随着新技术的不断更新和引入,供者池被不断扩大,比如使用年龄较大的供者、丙型肝炎病毒感染的供者、毒品过量致死的供者或心脏死亡器官捐献(DCD)供者的心脏等。与此同时,高龄、多器官功能不全、机械循环支持及人类白细胞抗原抗体致敏受者的比例近几年明显增加。供者数量的不足、受者状况的复杂化、免疫抑制治疗的个体化管理和远期移植物血管病的防治等都是心脏移植领域面临的挑战。本文通过概述现今全球在扩大供者库、提高受者质量、加强排斥反应的诊治和心脏移植物血管病变的预防等方面的新进展,以期有助于改善在等待或已经接受心脏移植的终末期心力衰竭患者的生存时间和生活质量。

常温机械灌注在无缺血肝移植中的应用体会

目的探讨常温机械灌注在无缺血肝移植手术中的应用体会。方法选取我院2017年7月23日~2017年8月25日应用常温机械灌注行无缺血肝移植的患者6例,术前做好人员准备、捐献供体准备、肝移植受体准备、环境和物品准备、器械和设备准备、机械灌注系统准备;术中配合医生完成无缺血供肝获取、无缺血供肝保存及无缺血肝移植手术。结果 6例供体肝脏均成功实施了无缺血器官获取术,手术时间3~4 h,平均3.5 h。器官机械灌注保存过程顺利,未发生异常。接着成功完成了6例全球首创不中断血流肝移植手术,手术过程顺利,手术时间5~7 h,平均6 h,手术出血600~2000 mL,平均1400 mL。结论常温机械灌注系统在无缺血肝移植手术中应用安全有效,术前准备充分,术中娴熟准确的护理配合是手术成功的基础和保障。

超冷器官保存技术及应用

在器官移植手术中,器官保存的主要目的是保持离体器官内部组织细胞的活性,为器官转运及分配、受者术前评估以及手术团队准备赢得时间。器官保存的主要方法可分为常温机械灌注保存和低温保存。其中低温保存是目前临床上最为常用的器官保存方法,但在低温保存状态下,组织细胞内部仍存在代谢活动,这导致器官的长时间保存极为困难。超冷器官保存技术是一种新型的低温器官保存技术,极大地延长了器官的保存时间,未来有望成为器官保存的重要手段,为“器官库”的建立提供技术支持。

他克莫司对肝移植急性排斥反应大鼠肠道菌群的影响

目的探讨他克莫司对心脏死亡器官捐献(donation after cardiac death,DCD)供肝移植后急性排斥反应大鼠肠道菌群的影响。方法构建稳定运行的常温机械灌注(normothermic machine perfusion,NMP)系统。夹闭Lewis大鼠胸主动脉30 min获取DCD肝脏,以棕色挪威大鼠(BN)为受体,建立原位肝移植急性排斥反应模型。根据对肝脏的不同处理方法,将BN大鼠分为以下3组:假手术(Sham)组、单纯NMP(NMP)组及NMP联合术后他克莫司(FMP)组,每组6只,术后14 d收集血液、肝脏和肠道内容物样本。血液标本检测肝功能,HE染色观察肝脏组织学变化并计算排斥活动指数(rejection activity index,RAI),16 SrDNA检测肠道内容物,观察各组肠道菌群结构、多样性及功能变化。结果FMP组(中位生存期>60 d)大鼠存活时间较NMP组(中位生存期为16.5 d)延长,肝功能和肝组织病理较NMP组显著改善(P<0.05)。计算RAI结果显示,FMP组(2.00±1.23)显著低于NMP组(7.60±1.14)(P<0.05)。与NMP组相比,FMP组肠道菌群丰度下降,两组在物种之间有明显差异,FMP组富含厚壁菌门、阿克曼菌科和毛螺菌科,且富集其他聚糖降解和鞘脂代谢通路。结论他克莫司能够改善DCD供肝移植后急性排斥反应大鼠肝损伤,同时调节肠道菌群结构和代谢途径。