Retrograde flush following topical cooling is superior to preserve the non-heart-beating donor lung

Objective: The use of non-heart-beating donors (NHBD) has been propagated as an alternative to overcome the scarcity of pulmonary grafts. Formation of microthrombi after circulatory arrest, however, is a major concern for the development of reperfusion injury. We looked at the effect and the best route of pulmonary flush following topical cooling in NHBD. Methods: Non-heparinized pigs were sacrificed by ventricular fibrillation and divided into three groups (n = 6 per group). After 1 h of in situ warm ischaemia and 2.5 h of topical cooling, lungs in group I were retrieved unflushed (NF). In group II, lungs were explanted following an anterograde flush (AF) through the pulmonary artery with 50 ml/kg Perfadex^® (6 °C). Finally, in group III, lungs were retrieved after an identical but retrograde flush (RF) via the left atrium. Flush effluent was sampled at intervals to measure haemoglobin concentration. Performance of the left lung was assessed during 60 min in our ex vivo reperfusion model. Wet-to-dry weight ratio (W/D) of both lungs was calculated as an index of pulmonary oedema. IL-1ß and TNF- protein levels in bronchial lavage fluid from both lungs were compared between groups. Results: Haemoglobin concentration (g/dl) was higher in the first effluent in RF versus AF (3.4 ± 1.1 vs 0.6 ± 0.1; p < 0.05). Pulmonary vascular resistance (dynes × s × cm⁻⁵) was 975 ± 85 RF versus 1567 ± 98 AF and 1576 ± 88 NF at 60 min of reperfusion (p < 0.001). Oxygenation (mmHg) and compliance (ml/cmH₂O) were higher (491 ± 44 vs 472 ± 61 and 430 ± 33 NS, 22 ± 3 vs 19 ± 3 and 14 ± 1 NS, respectively) and plateau airway pressure (cmH₂O) was lower (11 ± 1 vs 13 ± 1 and 13 ± 1 NS) after RF versus AF and NF, respectively. No differences in cytokine levels or in W/D ratios were observed between groups after reperfusion. Histology demonstrated microthrombi more often present after AF and NF compared to RF. Conclusion: Retrograde flush of the lung following topical cooling in the NHBD results in a better washout of residual blood and microthrombi and subsequent reduced pulmonary vascular resistance upon reperfusion.     

部分液体通气对无心跳供体肺的保护作用

目的 观察部分液体通气对无心跳供体(NHBD)肺的保护作用.方法 36只清洁级SD大鼠随机分为3组:氧气组(C组)、盐水组(B组)和部分液体通气组(A组).建立NHBD肺模型后行机械通气2h并监测大鼠气道压,2h后测量P-V曲线,切除大鼠左肺下叶,观察供体肺病理特点.行右肺支气管灌洗,测量灌洗液中肺表面活性物质相关蛋白B、C(SP-B、SP-C)的浓度.切除大鼠右肺上叶,并进一步行SP-B、SP-C的实时定量聚合酶链反应(Real-time PCR)检测.结果 A组大鼠动态顺应性和静态顺应性在2h热缺血期间都优于B和C组(P＜0.05);病理组织评分A组优于B组和C组(P＜0.05);肺泡灌洗液酶联免疫吸附试验(ELISA)检测和肺组织的Real-time PCR检测说明SP-B和SP-C蛋白和RNA含量差异有统计学意义,其中A组含量明显高于B组和C组(P＜0.05).结论 部分液体通气可以对热缺血期间供肺的肺功能和肺组织起到很好的保护作用,并对SP-B和SP-C有较好的保护作用.     

心脏停跳大鼠肝脏过氧化亚硝酸介导的细胞凋亡变化

目的 该实验旨在研究过氧化亚硝酸对心脏停跳大鼠肝细胞凋亡的影响.方法 切取雄性Wistar大鼠肝脏后,通过门静脉插管灌注60 ml 4℃HTK保存液,其中心脏搏动组为立即插管;心脏停跳组为60 min后插管;另外一组为心脏停跳但灌注液中含有7500 IU超氧化物歧化酶(SOD).(n均=6).然后所有的大鼠肝脏均在4℃HTK液中保存24 h后置于循环灌注系统中,用Kreb-Henseleit缓冲液常温再灌注45 min.结果 同心脏停跳组相比,心脏搏动和SOD组大鼠灌注液中转氨酶(GPT和GLDH)的含量明显减少(均P<0.05);门静脉压力明显降低(均P<0.01);胆汁分泌量明显增加(均P<0.01),一氧化氮含量明显降低(均P<0.05);细胞凋亡明显减少(均P<0.05);灌洗液中硝基酪氨酸明显减少(P<0.01和P<0.05)且硝基酪氨酸免疫组化染色明显减弱.结论 氧自由基的代谢产物--过氧化亚硝酸与心脏停跳大鼠肝脏细胞凋亡的发生相关.     

Kidney transplantation from non-heart-beating donors after oxygenated low-flow machine per fusion preservation with histidine-tryptophan-ketoglutarate solution

Abstract The aim of this study was to determine the potential benefit of aerobic machine preservation (MP) with non‐colloidal histidine‐tryptophan‐ketoglutarate (HTK) solution compared with MP with Belzer machine perfusion solution (MPS) and standard cold storage, after marginal kidneys had been obtained from non‐heart‐beating donors. Cardiac arrest was electrically induced in anaesthetized German landrace pigs (20–25 kg bw). Their kidneys were harvested 40 min thereafter, flushed with HTK by gravity of 100 cm H₂O via the renal artery and then stored in HTK for 18 h at 4°C. Other organs were subjected to oxygenated (pO2 > 500 mmHg) hypothermic pulsatile low‐flow machine perfusion with HTK or MP with Belzer MPS at Pmax = 40 mmHg, yielding trans‐renal flow values of 0.2–0.3 ml/min per g with HTK and approximately twice that amount with Belzer MPS. A well‐preserved vascular endothe‐lium and intact tubular epithelium were documented by electron microscopy at the end of perfusion preservation in both solutions as well as after cold storage. Concentrations of ATP (in micromoles per gramme) in tissue homogenates at the end of perfusion preservation with HTK were 1.18±0.12 vs 0.16 ± 0.02 (P>0.05) after simple cold storage and 2.43 ±0.23 after perfusion with Belzer MPS, thus documenting a relevant effect of low‐flow perfusion on tissue oxygenation. Viability of the grafts was followed for 1 week after heterotopic transplantation and bilateral ne‐phrectomy in the recipient pigs. Machine perfusion with HTK significantly improved cortical micro‐circulation upon early reperfusion in vivo, as well as maximal serum levels of urea and creatinine, compared to recipients receiving cold‐stored grafts. No differences could be found between MP with HTK or Belzer MPS. In conclusion, provision of oxygen during storage is possible by low‐flow perfusion with HTK as with Belzer MPS and apparently improves graft viability after transplantation.     

双层法氧合冷保存心跳停搏大鼠肝细胞移植研究

目的 观察双层法(TLM)氧合冷保存较UW保存能否改善心跳停搏供体(NHBD)肝细胞存活率和功能.方法 SD大鼠为供体,建立NHBD模型,NAPs大鼠为受体.根据热缺血时间(WIT)15 m/n和30 m/n分成2组;按TLM、UW分别保存3、12 h和未保存再各分5个亚组(n=5).检测NHBD肝细胞存活率和ATP水平,观察肝细胞移植(HTx)后肝细胞形态和功能.结果 TLM3、12 h组肝细胞存活率分别显著高于UW 3、12 h组[(69.7±4.1)%和(69.1±2.0)%比(55.1±2.3)%和(53.3±2.0)%;P<0.01];TLM 3、12 h组AlP水平分别显著高于UW 3、12 h组(3.25±0.79和3.06±0.67比2.25±0.53和1.63±0.40;P<0.05或P<0.01).HTx后几乎所有时间点TLM组血清白蛋白(ALB)水平都显著高于UW组(P<0.05或P<0.01).在HTx 14d后,形态学显示TLM组肝细胞保持强活力,糖原和ALB染色呈强阳性.结论 TLM氧合冷保存可显著改善和逆转NHBD肝细胞存活率和功能,减少NHBD肝细胞缺血性损伤.     

Normothermic recirculation reduces primary graft dysfunction of kidneys obtained from non-heart-beating donors

Our aim was to analyze the short- and long-term function of kidneys procured from non- heart-beating donors (NHBD) by means of three techniques: in situ perfusion (ISP), total body cooling (TBC) and normothermic recirculation (NR). Fifty-seven potential NHBD were included. Mean warm ischemia time was 68.9 ± 35.6 min. Forty-four kidneys were obtained from donors perfused with ISP, 8 with TBC, and 8 with NR. Eighteen kidneys (32 %) started functioning immediately, 29 (52 %) showed delayed graft function (DGF) and 9 (16 %) showed primary non function (PNF). The actuarial graft survival rate was 76.4 % at 1 year and 56 % at 5 years. The patient survival rate was 89.3 % at 5 years. Incidence of DGF and PNF was significantly lower in kidneys perfused with NR than those with ISP or TBC (P < 0.01). Duration of DGF was shorter in kidneys obtained through TBC than in kidneys obtained with ISP (P < 0.05). In conclusion, NR reduces the incidence of DGF and may be considered the method of choice for kidney procurement from NHBD. Received: 21 July 1999 Revised: 11 January 2000 Accepted: 5 May 2000     

大鼠心跳停搏供肝在原位肝移植术中损伤的预防

目的：探讨预防和减轻大鼠心跳停搏供肝在原位肝移植术中的损伤，以提高手术成功率。方法：雄性SD大鼠随机分为心跳停搏热缺血30min(N-30)和45min(N-45)两组;，每组分别行原位肝移植术30只次。同时，根据是否对供体手术方法进行改进又分为常规组和改良组。结果：(1)常规组和改良组的冷缺血时间分别为(70.04±1.48)和(70.36±1.42)min(P>0.05)，无肝期均为(16.40±0.73)min，肝下下腔静脉阻断时间均为(22.75±1.16)min，受体手术时间均为(90.58±3.76)min。(2)N-30和N-45常规组分别有5和9只受体术后死于原发性移植肝无功能，而改良组仅为1和2只(40%∶12%,P<0.05);(3)N-30和N-45组因术中分别出现供肝损伤致再灌注后供肝大量渗血、无肝期过长、切除受体肝脏时麻醉过深，而各有5和7，2和1，2和2只受体术后死亡。(4)N-30和N-45组术后1周存活率分别为50%和30%(P<0.05)。结论：预防心跳停搏供肝游离时损伤、供肝再灌注后渗血、无肝期过长和切除受体肝脏时麻醉过深是大鼠心跳停搏供肝原位肝移植手术成功的关键。     

便携式在体机械灌注设备运行稳定性及保护无心跳供体器官的实验研究

目的探讨便携式在体机械灌注设备(IMPD)运行稳定性和保护无心跳大动物供器官的作用。 方法6只健康清洁巴马小型猪麻醉后开腹，分别经腹主动脉、下腔静脉插管并连接到自主研发的便携式IMPD，静脉注射氯化钾骤停心脏，观察5 min无复跳，启动设备对动物器官进行常温(37 ℃)机械灌注。调整离心泵转速和灌注流量以控制静脉血氧饱和度维持在60%～70%，持续灌注12 h，每2小时为观察时间点，记录设备管路流量、离心泵转速和流量/转速比值，检测肝、肾功能和血液内环境指标，灌注结束后获取胆总管、肝、肾和小肠组织，HE染色后光镜下评估病理改变情况。采用重复测量资料单因素方差分析比较灌注过程中各时间点运行参数、肝肾功能和血液内环境指标，组内两两比较采用LSD法，P<0.05为差异有统计学意义。 结果全部实验均完成12 h灌注，灌注过程中设备运行稳定，无故障或停机意外，管路流量、离心泵转速和流量/转速比值均稳定。ALT、总胆红素(TBIL)和谷氨酰转移酶(GGT)稳定，灌注开始至结束各时间点与术前相比，差异均无统计学意义(P均>0.05)。灌注开始后AST缓慢上升，灌注第8、10和12小时AST分别为(73±21)、(90±30)和(114±48)(U/L)，与术前相比，差异均有统计学意义(P均<0.05)。在实验猪心脏停跳的5 min内，乳酸在灌注开始时便处于峰值，自灌注第8小时起，乳酸数值与峰值相比差异均有统计学意义(P均<0.05)。K^＋浓度水平自灌注第2小时起各时间点与灌注开始时相比，差异均有统计学意义(P均<0.05)。ALT、TBIL、GGT、血尿素氮、血清肌酐、血气分析指标、Na^＋、Ca^2＋和Cl^-浓度水平稳定，灌注开始至结束各时间点数值与灌注开始时相比，差异均无统计学意义(P均>0.05)。自灌注开始后持续有胆汁生成、尿量产生。供肝灌注12 h的过程中，肝脏始终保持外观红润、质地柔软、边缘锐利。灌注结束后，胆总管、肝、肾和小肠组织病理结果示组织结构完整，无淤血和大面积坏死征象。 结论便携式IMPD可以稳定灌注无心跳猪供器官12 h，可以较好维护胆总管、肝、肾和小肠器官。     

Kidney transplantation from non-heart-beating donors after oxygenated low-flow machine perfusion preservation with histidine–tryptophan–ketoglutarate solution

The aim of this study was to determine the potential benefit of aerobic machine preservation (MP) with non-colloidal histidine–tryptophan–ketoglutarate (HTK) solution compared with MP with Belzer machine perfusion solution (MPS) and standard cold storage, after marginal kidneys had been obtained from non-heart-beating donors. Cardiac arrest was electrically induced in anaesthetized German landrace pigs (20–25 kg bw). Their kidneys were harvested 40 min thereafter, flushed with HTK by gravity of 100 cm H₂O via the renal artery and then stored in HTK for 18 h at 4°C. Other organs were subjected to oxygenated (pO₂>500 mmHg) hypothermic pulsatile low-flow machine perfusion with HTK or MP with Belzer MPS at P_max=40 mmHg, yielding transrenal flow values of 0.2–0.3 ml/min per g with HTK and approximately twice that amount with Belzer MPS. A well-preserved vascular endothelium and intact tubular epithelium were documented by electron microscopy at the end of perfusion preservation in both solutions as well as after cold storage. Concentrations of ATP (in micromoles per gramme) in tissue homogenates at the end of perfusion preservation with HTK were 1.18±0.12 vs 0.16±0.02 (P<0.05) after simple cold storage and 2.43±0.23 after perfusion with Belzer MPS, thus documenting a relevant effect of low-flow perfusion on tissue oxygenation. Viability of the grafts was followed for 1 week after heterotopic transplantation and bilateral nephrectomy in the recipient pigs. Machine perfusion with HTK significantly improved cortical microcirculation upon early reperfusion in vivo, as well as maximal serum levels of urea and creatinine, compared to recipients receiving cold-stored grafts. No differences could be found between MP with HTK or Belzer MPS. In conclusion, provision of oxygen during storage is possible by low-flow perfusion with HTK as with Belzer MPS and apparently improves graft viability after transplantation.     

Machine perfusion for kidneys: how to do it at minimal cost

Due to a shortage of organs for transplantation, many centres use marginal grafts to increase their donor pool. As kidneys from non-heart-beating donors (NHBD) have sustained initial ischaemic damage, their viability is difficult to predict. Hypothermic pulsatile perfusion has not only been used to improve the condition of such grafts, but also allows viability assessment. Suitable systems are becoming more readily available, but they are expensive. We have used existing dialysis equipment with modified sterilised inserts to create a pulsatile hypothermic perfusion system. With this system, 41 NHBD kidneys were perfused for up to 8 h; their intravascular renal resistance (IRVR), flow characteristics as well as glutathione S transferase (GST) measurements were performed to assess viability. This hypothermic pulsatile perfusion system is now an integral component of our NHBD programme. Received: 11 January 2000 Revised: 13 June 2000 Accepted: 7 November 2000