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。     

The functional integrity of a normothermic perfusion system using artificial blood in pig liver

BACKGROUND: We have reported already that we succeeded in developing a normothermic liver perfusion system consisting of purely artificial products such as artificial blood. The aim of this study was to ascertain the metabolic functional integrity of the liver perfused in this system. MATERIALS AND METHODS: A liver graft from a female pig weighing 20 kg was harvested in the usual manner. The perfusion solution consisted of artificial blood, L-15 medium, distilled water, bovine serum albumin, NaHCO3, NaOH, KCl, human regular insulin, 50% glucose solution, and dexamethasone. The isolated liver was perfused with this oxygenated perfusate through the portal vein at a rate of 300 ml/min for 9 h. We analyzed the changes of amino acids, ammonia, and urea concentrations in five livers, which showed high oxygen consumption (over 8 ml-O2/min during perfusion). RESULTS: Although urea did not exist in the perfusate at first, an extremely high level of the urea concentration appeared during the perfusion. On the other hand, the ammonia concentration was only slightly elevated during the perfusion. Arginine vanished from the perfusate with increases of citrulline and ornithine. Taurine was elevated with the disappearance of methionine. Alanine, glutamine, serine, histidine, and threonine concentrations decreased with an increase of the glucose concentration. Numerous liver cells exhibited PAS-positive cytoplasmic glycogen deposits not exhibited before perfusion. The ratios of branched-chain amino acids/aromatic amino acids were elevated during the perfusion. CONCLUSION: Normothermic liver perfusion using artificial blood could sufficiently maintain the functional integrity of the liver.     

Role of oxygen during hypothermic machine perfusion preservation of the liver

Grafts from non‐heart‐beating donors are thought to be best preserved by hypothermic machine perfusion (HMP). Controversy exists concerning the role of oxygenation during HMP. In this study, we wanted to evaluate the relative role of oxygenation for graft integrity during and after HMP. Cardiac arrest was induced in male Wistar rats (250–300 g) by phrenotomy. Thirty minutes later, livers were flushed via the portal vein and subjected to 18 h of HMP at 5 ml/min at 4 °C. During HMP, the preservation solution was equilibrated with 100% oxygen (HMP100), with air (HMP20) or not oxygenated at all (HMP0). Graft integrity was assessed thereafter upon warm reperfusion in vitro. During preservation, oxygenation of the perfusate reduced alanine aminotransferase release by 50% compared with HMP0. HMP100 resulted in reduced oxygen free radical‐mediated lipid peroxidation upon warm reperfusion compared with both HMP20 and HMP0. One hundred per cent oxygenation during HMP also significantly enhanced the activation of AMPK salvage pathway, and upstream activation of protein kinase A when compared with HMP0. Enzyme release during reperfusion was reduced by approximately 40% (HMP20) or approximately 70% (HMP100) after oxygenation compared with HMP0. Functional recovery (bile production) was only enhanced by HMP100 (approximately twofold increase vs. HMP20 and HMP0, P < 0.05). Efficiency of HMP might be markedly increased by additional aeration of the perfusate, most successfully by equilibration with 100% oxygen.     

BMMSCs对SD大鼠DCD供肝常温机械灌注脂肪变性的影响机制研究

目的:研究铁死亡在骨髓间充质干细胞(BMMSCs)联合常温机械灌注(NMP)修复SD大鼠心脏死亡器官捐献(DCD)脂肪变性供肝中的作用。方法:提取SD大鼠BMMSCs。建立大鼠脂肪肝DCD模型。24只SD大鼠随机分为单纯脂肪肝组(Sham)、静态冷保存组(SCS)、常温机械灌注保存组(NMP)、BMMSCs联合NMP保...     

常温机械灌注修复边缘性供肝的临床疗效：单中心报道

目的探讨常温机械灌注（normothermic machine perfusion, NMP）修复边缘性供肝的安全性和有效性。 方法2018年9月至2019年9月,使用NMP进行6例边缘性供肝体外评估和修复,供肝来自4例心死亡和2例高胆红素血症患者。记录灌注过程的灌注参数、灌注液血气分析及生化检验指标,结合供肝外观等评估边缘性供肝是否适合移植。术后随访至少3个月,记录移植后7 d内肝功能指标、生化指标、并发症发生情况等。 结果NMP期间灌注参数稳定,肝动脉灌注流量为110~334 ml/min,门静脉灌注流量为540~1 180 ml/min。灌注液pH、PO₂、PCO₂在灌注0.5 h后基本恢复正常,乳酸水平迅速下降;肝酶水平无明显升高,胆汁pH>7.5。所有供肝灌注均匀,质地柔软,均被用于移植。受者肝移植后恢复情况良好,无一例发生原发性移植肝无功能或缺血性胆道病变,至末次随访所有患者及移植物均存活。 结论本科室在临床肝移植中应用NMP技术的初步经验提示,NMP用于边缘性供肝的保存是安全、有效的。     

Magnetic resonance imaging assessment of renal flow distribution patterns during ex vivo normothermic machine perfusion in porcine and human kidneys

Acceptance criteria of deceased donor organs have gradually been extended toward suboptimal quality, posing an urgent need for more objective pre-transplant organ assessment. Ex vivo normothermic machine perfusion (NMP) combined with magnetic resonance imaging (MRI) could assist clinicians in deciding whether a donor kidney is suitable for transplantation. Aim of this study was to characterize the regional distribution of perfusate flow during NMP, to better understand how ex vivo kidney assessment protocols should eventually be designed. Nine porcine and 4 human discarded kidneys underwent 3 h of NMP in an MRI-compatible perfusion setup. Arterial spin labeling scans were performed every 15 min, resulting in perfusion-weighted images that visualize intrarenal flow distribution. At the start of NMP, all kidneys were mainly centrally perfused and it took time for the outer cortex to reach its physiological dominant perfusion state. Calculated corticomedullary ratios based on the perfusion maps reached a physiological range comparable to in vivo observations, but only after 1 to 2 h after the start of NMP. Before that, the functionally important renal cortex appeared severely underperfused. Our findings suggest that early functional NMP quality assessment markers may not reflect actual physiology and should therefore be interpreted with caution.     

常温机械灌注修复长时间冷保存供肝的大动物实验研究

目的探索常温机械灌注(NMP)在挽救大动物边缘供肝中的价值。 方法6只雄性、10～12月龄巴马小型猪分为静态冷保存(SCS)6 h组和SCS 24 h组,每组3只,分别于供肝获取后SCS 6 h、24 h后进行2 h NMP复苏。基于荷兰Organ Assist公司Liver Assist系统和供体猪自身血液,整合四通道生理仪器及自制灌注管路搭建NMP平台。在NMP过程中分别于灌注0、15、60、90、120 min 5个时间点收集灌注液用于肝功能(ALT、AST)检测及血气分析(pH、氧分压)。灌注结束取肝左叶相同位置少许组织,以10%甲醛固定,用于后续HE染色。 结果NMP开始时,SCS 24 h组ALT、AST水平略高于SCS 6 h组,之后AST、ALT水平均缓慢上升。NMP开始时SCS 24 h组pH值中位数为7.28,氧分压中位数为46 mmHg(1 mmHg＝0.133 kPa,下同),均低于SCS 6 h组(7.36,52 mmHg),经过2 h NMP后SCS 24 h组pH值和氧分压逐渐接近甚至优于SCS 6 h组。HE染色发现,灌注前SCS 24 h组肝脏充血严重,炎性细胞浸润明显;NMP 2 h后,肝脏充血明显改善、炎性细胞减少,与SCS 6 h组无明显差异。 结论基于Liver Assist系统和供体猪自身血液成功搭建的NMP平台,可有效改善长时间冷保存的边缘供肝质量,提示NMP在修复边缘供肝、扩展供肝来源方面具有重要临床及基础研究价值。     

Role of perfusion medium, oxygen and rheology for endoplasmic reticulum stress-induced cell death after hypothermic machine preservation of the liver.

Recently, the endoplasmic reticulum (ER) has been disclosed as subcellular target reactive to ischaemia/reperfusion and possibly influenced by hypothermic machine preservation. Here, the respective role of perfusate, perfusion itself, and the effect of continuous oxygenation to trigger ER-stress in the graft should be investigated. Livers were retrieved 30 min after cardiac arrest of male Wistar rats and preserved by cold storage (CS) in histidine-tryptophan-ketoglutarate (HTK) for 18 h at 4 degrees C. Other organs were subjected to aerobic conditions either by oxygenated machine perfusion with HTK (MP-HTK) or Belzer solution (MP-Belzer) at 4 degrees C or by venous insufflation of gaseous oxygen during cold storage (VSOP). Viability of livers was evaluated upon reperfusion in vitro according to previously validated techniques for 120 min at 37 degrees C. Oxygenation during preservation (MP-HTK, MP-Belzer or VSOP) concordantly improved functional recovery (bile flow, ammonia clearance), reduced parenchymal enzyme leakage and histological signs of necrosis and significantly attenuated mitochondrial induction of apoptosis (cleavage of caspase 9) compared to CS. However, MP with either medium produced about 500% elevated protein expression of CHOP/GADD153, suggesting pro-apoptotic ER-stress responses, paralleled by a significant elevation of caspase-12 enzyme activity compared to CS or VSOP. Although MP also promoted a slight (20%) induction of the cytoprotective ER-protein Bax inhibitor protein (BI-1), prevailing of proapoptotic reactions was seen by increased cleavage of caspase-3 and poly (ADP-Ribase)-polymerase (PARP) in both MP-groups. Endoplasmic stress activation is conjectured a specific side effect of long-term machine preservation irrespective of the medium, actually promoting cellular apoptosis via activation of caspase-12. The simple insufflation of gaseous O2 may be considered a feasible alternative, apparently indifferent to the endoplasmic reticulum.     

Pretransplant sequential hypo‐ and normothermic machine perfusion of suboptimal livers donated after circulatory death using a hemoglobin‐based oxygen carrier perfusion solution

Ex situ dual hypothermic oxygenated machine perfusion (DHOPE) and normothermic machine perfusion (NMP) of donor livers may have a complementary effect when applied sequentially. While DHOPE resuscitates the mitochondria and increases hepatic adenosine triphosphate (ATP) content, NMP enables hepatobiliary viability assessment prior to transplantation. In contrast to DHOPE, NMP requires a perfusion solution with an oxygen carrier, for which red blood cells (RBC) have been used in most series. RBC, however, have limitations and cannot be used cold. We, therefore, established a protocol of sequential DHOPE, controlled oxygenated rewarming (COR), and NMP using a new hemoglobin‐based oxygen carrier (HBOC)‐based perfusion fluid (DHOPE‐COR‐NMP trial, NTR5972). Seven livers from donation after circulatory death (DCD) donors, which were initially declined for transplantation nationwide, underwent DHOPE‐COR‐NMP. Livers were considered transplantable if perfusate pH and lactate normalized, bile production was ≥10 mL and biliary pH > 7.45 within 150 minutes of NMP. Based on these criteria five livers were transplanted. The primary endpoint, 3‐month graft survival, was a 100%. In conclusion, sequential DHOPE‐COR‐NMP using an HBOC‐based perfusion fluid offers a novel method of liver machine perfusion for combined resuscitation and viability testing of suboptimal livers prior to transplantation.     

Human liver stem cell-derived extracellular vesicles reduce injury in a model of normothermic machine perfusion of rat livers previously exposed to a prolonged warm ischemia

Livers from donors after circulatory death (DCD) are a promising option to increase the donor pool, but their use is associated with higher complication rate and inferior graft survival. Normothermic machine perfusion (NMP) keeps the graft at 37°C, providing nutrients and oxygen supply. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) are able to reduce liver injury and promote regeneration. We investigated the efficacy of a reconditioning strategy with HLSC-EVs in an experimental model of NMP. Following total hepatectomy, rat livers were divided into 4 groups: (i) healthy livers, (ii) warm ischemic livers (60 min of warm ischemia), (iii) warm ischemic livers treated with 5 × 10⁸ HLSC-EVs/g-liver, and (iv) warm ischemic livers treated with a 25 × 10⁸ HLSC-EVs/g-liver. NMP lasted 6 h and HLSC-EVs (Unicyte AG, Germany) were administered within the first 15 min. Compared to controls, HLSC-EV treatment significantly reduced transaminases release. Moreover, HLSC-EVs enhanced liver metabolism by promoting phosphate utilization and pH self-regulation. As compared to controls, the higher dose of HLSC-EV was associated with significantly higher bile production and lower intrahepatic resistance. Histologically, this group showed reduced necrosis and enhanced proliferation. In conclusion, HLSC-EV treatment during NMP was feasible and effective in reducing injury in a DCD model with prolonged warm ischemia.     

Synthetic hemoglobin‐based oxygen carriers are an acceptable alternative for packed red blood cells in normothermic kidney perfusion

Normothermic machine perfusion presents a novel platform for pretransplant assessment and reconditioning of kidney grafts. Maintaining the metabolic activity of a preserved graft at physiologic levels requires an adequate oxygen supply, typically delivered by crystalloid solutions supplemented with red blood cells. In this study, we explored the feasibility of using a synthetic hemoglobin‐based oxygen carrier (HBOC) in human kidney normothermic perfusion. Fourteen discarded human kidneys were perfused for 6 hours at a mean temperature of 37°C using a pressure‐controlled system. Kidneys were perfused with a perfusion solution supplemented with either HBOC (n = 7) or packed red blood cells (PRBC) (n = 7) to increase oxygen‐carrying capacity. Renal artery resistance, oxygen extraction, metabolic activity, energy stores, and histological features were evaluated. Throughout perfusion, kidneys from both groups exhibited comparable behavior regarding vascular flow (P = .66), oxygen consumption (P = .88), and reconstitution of tissue adenosine triphosphate (P = .057). Lactic acid levels were significantly higher in kidneys perfused with PRBC (P = .007). Histological findings were comparable between groups, and there was no evidence of histological damage caused by the HBOC. This feasibility experiment demonstrates that a HBOC solution can offer a logistically more convenient off‐the‐shelf alternative to PRBC in normothermic machine perfusion of human kidneys.     

Effect of normothermic perfusion using fructose-1,6-bisphosphate for maintenance of liver function during in situ extended hepatectomy by the total hepatic vascular exclusion technique

BACKGROUND: Recently, hepatic surgery has made remarkable progress, and it is important to use appropriate liver perfusion. We evaluated the effect of normothermic liver perfusion with the addition of fructose-1, 6-bisphosphate (FBP) and oxygenation to maintain liver parenchymal, non-parenchymal, and Kupffer cell function. MATERIALS AND METHODS: The rats were divided into five groups according to the perfusate and continuous perfusion was performed: Control group = 4 degrees C lactate Ringer with 10% glucose (LRG) solution; normothermic group = 25 degrees C LRG solution; normothermic oxygenated group = 25 degrees C oxygenated LRG solution; normothermic FBP group = 25 degrees C LRG solution with addition of 10 mmol/L FBP; normothermic oxygenated FBP group = 25 degrees C oxygenated LRG solution with addition of 10 mmol/L FBP. Parameters under evaluation were oxygen consumption, liver energy level (adenosine triphosphate, total adenine nucleotide), glutathione, lipid peroxide, hyaluronic acid uptake ratio, apoptosis, and histomorphology. Moreover, we studied the effect of FBP and normothermia on Kupffer cells activation in vitro. RESULTS: Liver energy level was lower in the normothermic group than the control group. But, it was improved by oxidation or addition of FBP, and it was satisfactorily maintained up to 120 min in the group with normothermic oxygenated FBP. Hyaluronic acid uptake was maintained highly at all times as measured in normothermic oxygenated FBP group. The uptake of lipopolysaccharide was significantly higher as a result of adding FBP, compared with that in the control group and the normothermic group. Moreover, the apoptotic index in the liver was decreased in normothermic FBP group compared to control group. CONCLUSIONS: The normothermic liver perfusion under additional FBP and oxygenation protects both parenchymal and non-parenchymal cells from reperfusion injury.     

低温双通道氧合机械灌注在离体劈离式肝移植中的应用

随着劈离式肝移植的飞速开展,其技术日趋成熟。离体劈离时,由于细胞内能量来源[如三磷酸腺苷(ATP)]的耗竭以及其他代谢紊乱,导致细胞损伤和功能障碍,这种损伤会因肝移植再灌注损伤而加重,临床上表现为移植术后并发症和移植失败。为了进一步改善离体劈离式肝移植的供肝质量,国内外研究团队使用机械灌注来改善供肝质量。本文结合了国际上离体劈离式肝移植供肝机械灌注的研究进展,阐述了低温双通道氧合机械灌注(D-HOPE)在离体劈离式肝移植中的应用和未来发展方向,旨在为增加离体劈离式肝移植的供肝来源,进一步改善供肝短缺的现状提供参考。     

Liver preservation with HTK: salutary effect of hypothermic aerobiosis by either gaseous oxygen or machine perfusion

The aim of the present study was to improve the viability of marginal livers from non-heart beating donors upon cold preservation using two different techniques for the provision of tissue aerobiosis. Livers from male Wistar rats (250-300 g bw) were harvested after 60 min of cardiac arrest, flushed via the portal vein with 20 mL of heparinized Ringer's solution and 60 mL of histidine-tryptophan-ketoglutarate (HTK) preservation solution. Control livers were then stored submerged in HTK for 24 h at 4 degrees C while other organs were subjected to aerobic conditions by either insufflation of gaseous oxygen via the venous vascular system of the cold stored organ (VSOP) or pulsatile machine perfusion (MP) with oxygenated HTK at 5 mL/min at 4 degrees C. Superoxide dismutase (SOD) (7500 IU) was added to the last 10 mL of HTK in order to prevent adverse effects of high oxygen tensions at hypothermia. Viability of the livers was assessed upon isolated perfusion in vitro with oxygenated Krebs-Henseleit buffer at constant flow. VSOP or MP, both significantly improved vascular conductivity upon reperfusion as evaluated by portal venous pressure, reduced hepatic enzyme release and led to a rise in hepatic bile production upon reperfusion. Induction of apoptosis was also looked for in tissue homogenates by Western analysis for cleavage of poly(ADP-ribose)polymerase (PARP). Expression of cleaved PARP fragment could be found in reperfused control livers but also, though to a lesser extend, after VSOP or MP. In conclusion, provision of oxygen during cold preservation significantly contributes to improve organ viability upon reperfusion and must be regarded as a useful adjunct for marginal or pre-damaged livers. HTK has been shown for the first time to be also suitable for long-term MP preservation of the liver, but, as inferred from these data, simple insufflation of gaseous O2 may be considered a feasible alternative.     

Predictor parameters of liver viability during porcine normothermic ex situ liver perfusion in a model of liver transplantation with marginal grafts

Normothermic ex situ liver perfusion (NEsLP) offers the opportunity to assess biomarkers of graft function and injury. We investigated NEsLP parameters (biomarkers and markers) for the assessment of liver viability in a porcine transplantation model. Grafts from heart‐beating donors (HBD), and from donors with 30 minutes (donation after cardiac death [DCD]30′), 70 minutes (DCD70′), and 120 minutes (DCD120′) of warm ischemia were studied. The HBD, DCD30′, and DCD70′‐groups had 100% survival. In contrast, 70% developed primary nonfunction (PNF) and died in the DCD120′‐group. Hepatocellular function during NEsLP showed low lactate (≤1.1 mmol/L) in all the groups except the DCD120′‐group (>2 mmol/L) at 4 hours of perfusion (P = .04). The fold‐urea increase was significantly lower in the DCD120′‐group (≤0.4) compared to the other groups (≥0.65) (P = .01). As for cholangiocyte function, bile/perfusate glucose ratio was significantly lower (<0.6) in all the groups except the DCD120′‐group (≥0.9) after 3 hours of perfusion (<0.01). Bile/perfusate Na+ ratio was significantly higher (≥1.2) after 3 hours of perfusion in all the groups except for the DCD120′‐group (≤1) (P < .01). Three hours after transplantation, the DCD120′‐group had a significantly higher international normalized ratio (>5) compared to the rest of the groups (≤1.9) (P = .02). Rocuronium levels were higher at all the time‐points in the animals that developed PNF during NEsLP and after transplantation. This study demonstrates that biomarkers and markers of hepatocellular and cholangiocyte function during NEsLP correlate with the degree of ischemic injury and posttransplant function.