The impact of liver preservation in HTK and UW solution on microcirculation after liver transplantation

Severe microcirculatory disturbances due to endothelial cell damage and leukocyte adherence during reperfusion of transplanted livers are considered to contribute to early graft failure. Since the degree of reperfusion injury after liver transplantation depends on the length of preservation time and the solution used for preservation, the aim of our study was to assess three solutions with respect to microvascular perfusion and leukocyte adhesion. Therefore, rat livers were stored up to 24 h in Euro-Collins (EC), University of Wisconsin (UW), or histidin-tryphtophan-ketoglutarate (HTK) solutions prior to orthotopic transplantation. The livers were studied in situ 60 min postoperatively using intravital fluorescence video microscopy. Using simple syringe flushing (10 ml), sinusoidal perfusion decreased below 50% in EC preserved livers after 8 h preservation, in HTK preserved livers after 16 h preservation, and remained higher than 70% in livers preserved in UW up to 24 h. Permanent adhesion of leukocytes was increased more rapidly in organs after 1, 8, 16, and 24 h preservation in HTK (16%, 15%, 34%, and 49.7% ± 4.7%) compared to those preserved in UW (15%, 18%, 17%; and 32.7% ± 3.3%; P < 0.05). Using a 10-fold volumn of the organ weight of HTK solution during the harvesting procedure, with an 8 min equilibration period, sinusoidal perfusion (39.6 ± 4.7%) and leukocyte adhesion (42.7 ± 3.1%) were not improved after 24 h. In contrast, equilibration with a volumn of approximately 40-times the liver weight improved sinusoidal perfusion (70.8% ± 2.7%; P < 0.01) and leukocyte adhesion (24.9% ± 3.1%; P < 0.01) significantly. Thus, using HTK solution, simple flushing prior to long-term cold storage resulted in microcirculatory disturbances when compared to UW solution. Larger volumns of HTK solution with an additional equilibration period of 8 min, however, reduced leukocyte adhesion and improved sinusoidal perfusion to a similar degree as UW solution.     

The addition of mood and anxiety domains to the University of Washington quality of life scale

BACKGROUND: There are numerous head and neck specific quality of life questionnaires, each having its own merits and disadvantages. The University of Washington questionnaire has been widely used and is notable by the inclusion of a shoulder dysfunction domain, domain importance ratings, and patient free text. It is short, simple to process, and provides clinically relevant information. However, it has lacked any psychological dimension of quality of life. The aim of this study was to report the inclusion of two psychological domains (mood, anxiety) to the most recent refinement of the questionnaire (version 3). METHOD: A cross-sectional survey was performed in April 2000. Questionnaires were sent to 183 patients alive and disease free after surgery for oral and oro-pharyngeal malignancy. Replies were received from 145 patients (79% response rate). RESULTS: The new domains (mood and anxiety) correlated significantly with the emotional functioning domains from the EORTC C30 and with the pain and appearance domains of UW-QOL. There were also significant correlations between the "global quality of life" item and the two new domains. Mood (p =.005) and anxiety (p <.001) scores were associated with patient age but with no other clinicodemographic variable. CONCLUSION: The addition of mood and anxiety domains makes the UW-QOL version 4 a single broad measure suitable for effective health-related quality of life evaluation in the routine clinical setting.     

31P-NMR study of cardiac preservation: St. Thomas' Hospital cardioplegic solution versus UW preservation solution

Ex vivo cardiac preservation was evaluated by measuring the catabolism of high-energy phosphate (ATP and creatine phosphate, CrP) using ³¹P-NMR spectroscopy. After cardioplegic arrest St. Thomas' Hospital cardioplegic solution (group A), and University of Wisconsin (UW) preservation solution (group B) were tested. The hearts were mounted in the 4.7 T horizontal bore magnet of the NMR spectrometer and were continuously perfused with the test solution under 25 cm H₂O pressure for 6 h at 10°C. Peak heights of the -phosphate of ATP and CrP were measured and expressed as percentages of the initial value. For both group A and group B, ATP declined less rapidly during preservation than CrP. In group A, ATP remained constant for 60 min while CrP decreased from the onset of preservation. After 6h of preservation 28.3% of ATP and 24.5% of CrP remained (group A). On the other hand, in group B, levels of both ATP and CrP remained much more stable: CrP did not decrease during the first 3 h of preservation, while ATP started to decrease after 5 h. At the end of preservation 76.1% of ATP and 71.5% of CrP were still present. We conclude that UW solution is superior to St. Thomas' Hospital solution for the preservation of high-energy phosphates during 6 h cardiac preservation with continuous hypothermic low-flow perfusion.     

13例原位心脏移植术中长时间心肌缺血的心肌保护和术后近期效果

目的 :总结分析 13例原位心脏移植手术中心肌缺血时间大于 2 4 0min的心肌保护技术和术后近期疗效。方法 2 0 0 0年 5月～ 2 0 0 4年 9月连续为 79例患者进行原位心脏移植术 ,其中 13例心肌缺血时间大于 2 4 0min。本组术前心功能均为Ⅳ级。手术按标准法行原位心脏移植手术 2例 ,行双腔静脉吻合法移植手术 11例。心肌缺血时间在 2 4 0～ 383min ,平均 (312± 4 2 )min。供心的保护方法包括在主动脉阻断时经主动脉根部灌注晶体停搏液 ,在取下心脏后 ,再次灌注UW液 ,并置于UW液中保存。结果 :本组无手术死亡。术后均获完整随访 ,随访时间 1个月～ 3年 ,平均 8.2个月 ,术后无死亡 ,所有病人心功能均恢复至I～II级。结论 :我们所采取的心脏获取和心肌保护方法 ,加之合适的供体、受体选择 ,双腔静脉吻合技术 ,以及加强术中、术后监测和处理 ,长时间心肌缺血的病人可获得满意的早期疗效。     

UW solution: a promising tool for cryopreservation of primarily isolated rat hepatocytes

Cryopreserved hepatocytes are a ready source of metabolic and synthetic functions for hepatocyte transplantation and bioartificial livers. In this study, we evaluated a cytoprotective effect of University of Wisconsin (UW) solution during cryopreservation of rat hepatocytes. We also investigated the feasibility of lentivirus-based gene transfer into thawed hepatocytes after cryopreservation. Primary rat hepatocytes were isolated using a two-step collagenase perfusion technique, and the resulting hepatocytes with more than 85% viability assessed by a trypan blue exclusion test were subjected to the present study. These cells were subjected to the present study. Cells were cryopreserved with UW solution containing 10% fetal bovine serum (FBS) with 12% dimethylsulfoxide (DMSO) (group 1, G1), Cellbanker solution (group 2, G2), and 10% FBS-containing Dulbecco modified Eagle medium (DMEM) with 12% DMSO (group 3, G3). After thawing the cryopreserved hepatocytes, cell viability, plating efficiency, morphological appearance, and ammonia clearance activity were determined for each group. The efficacy of lentivirus-mediated Escherichia coli LacZ gene delivery was evaluated. Hepatocyte viabilities after 3- and 7-day cryopreservation were 73.2% and 62.5% for G1, 57.5% and 46.5% for G2, and 57.3% and 41.5% for G3, respectively. Plating efficiency and ammonia clearance activity were improved in G1 hepatocytes compared to G2 and G3 cells. Lentiviral transfer of a LacZ gene was confirmed in the thawed hepatocytes after cryopreservation by an X-gal stain assay. Received: April 4, 2002 / Accepted: August 8, 2002 Acknowledgments. This work was supported in part by grants from the Ministry of Education, Science, and Culture, Japan, and the Ministry of Economy and Industry, Japan. Offprint requests to: J. Arikura     

长时间供心保护的原位心脏移植

目的总结3例供心离体保护时间较长的原位心脏移植过程和近期生存观察情况。方法2005年3～8月,先后对3例患有终末期扩张性心肌病患者（心功能均为Ⅳ级）行心脏移植手术;3例供体心脏离体保存时间分别为542、367、485mln。移植手术方式采用双腔静脉吻合法。供心的保护过程：主动脉根部灌注心脏停搏液,快速取下心脏后再灌注冷UW液,并放置于UW液中低温保存。结果3例移植手术均获成功,目前患者心功能在Ⅰ～Ⅱ级,生活质量好。结论合适的供体、快捷的心脏获取方法和心肌保存方法,在离体心脏保存时间较长时,合适的受体,并选用双腔静脉吻合技术,可收到良好手术效果。     

Protective strategies against ischemic injury of the liver

This article summarizes strategies to protect the liver from injuries caused by ischemia and reperfusion. Three different sections (i.e., surgical and pharmacologic strategies and gene therapy) present approaches to enhance the survival and viability of the liver in various surgical procedures including liver transplantation. The first section reviews approaches using surgical interventions such as ischemic preconditioning and intermittent clamping. Their protective effects are discussed with respect to the mechanism of injury. In the second section, pharmacologic agents targeting microcirculation, oxidative stress, proteases, and inflammation are described. Mechanisms of injury and their suppression by a wide variety of drugs are discussed. The third section focuses on gene therapy. Potential target genes have been identified (e.g., superoxide dismutase or heme oxygenase). Animal experiments in which the liver injury is reduced successfully may pave the way to novel strategies applied to different liver diseases in humans.     

新型胰岛分离液对小鼠胰岛分离效果的研究

目的  探讨新型胰岛分离液（IPS液）在小鼠胰岛分离中的分离效率及胰岛保护作用。方法  将消化后的小鼠胰腺按体积平均分为两组（IPS组和UW组）,分别应用IPS-Optiprep液及UW-Optiprep液进行连续梯度密度离心分离胰岛,比较两组分离液的分离纯化效率和分离后的胰岛活性。将诱导成功的糖尿病小鼠随机分为3组：实验组（n=10）,接受采用IPS-Optiprep液分离纯化的胰岛移植;对照组（n=10）,接受采用UW-Optiprep液分离纯化的胰岛移植;假性移植组（n=5）,仅给予手术但并不进行胰岛移植。分析3组小鼠术后血糖水平以及测实验组和对照组小鼠术后21 d的腹腔葡萄糖耐量试验的血糖水平。比较两种分离液的配制成本。结果  与UW组相比,IPS组的IPS-Optiprep液可提供更高的胰岛当量（IEQ）、胰岛纯度、回收率及胰岛完整度。胰岛形态观察可见,IPS组胰岛被膜完整,直径明显大于UW组。UW组纯化后的胰岛活性率高于IPS组[（88±5）%比（84±3）%,P < 0.01]。与UW-Optiprep液相比,IPS-Optiprep液可获得相当的在体胰岛功能。IPS-Optiprep液可显著降低胰岛分离纯化成本。结论  新型胰岛分离液IPS-Optiprep液在胰岛分离提纯中显示出较好的分离效率,增加了胰岛的纯度、完整度与回收率,并显著降低了纯化成本,但对胰岛细胞活性的保护作用稍逊,可能与胰岛的高完整度及IPS-Optiprep液中的内毒素有关。     

Comparison of University of Wisconsin and University of Pittsburgh solutions for heart transplantation

The effectiveness of University of Wisconsin (UW) and University of Pittsburgh (UP) solutions for the preservation of rat hearts was compared. Lewis rat hearts were preserved with UW (group A, n=45) or UP (group B, n=45) solution for 0 or 24 h and then transplanted heterotopically into the recipients' abdomen. Ten recipients in each group were observed to obtain 1-week graft survival rates. Tissue water content and tissue content of adenine nucleotides were measured 2 h after transplantation in six grafts from each group. Six hearts preserved for 0 h and seven hearts preserved for 24 h were taken from each group 24 h after grafting for histopathology. The 1-week graft survival rates of groups A24 and B24 were 60% and 10%, respectively. In the 24-h preserved grafts, adenosine triphosphate (ATP) and energy charge [(ATP+adenosine diphosphate/2)/(ATP+adenosine diphosphate+adenosine monophosphate)] of groups A and B were 0.972±0.165 and 0.200±0.123 mg/g wet tissue (P<0.05) and 74.4% and 61.1% (P<0.05), respectively. The tissue water content of group A24 was 71.7%, whereas that of group B24 was 74.1% (P<0.05). Histopathology revealed more severe muscle edema and necrosis and infiltration of polymorphonuclear cells in group B24 than in group A24. We conclude that UW solution is more appropriate for rat heart preservation than UP solution.     

Effect of different preservation solutions on adenine nucleotide content and metabolism in human kidney transplantation

Differences in purine metabolism produced by three preservation solutions were studied by determining the adenine nucleotide (ATP, ADP, AMP, and IMP) and nucleoside (adenosine, inosine, and hypoxanthine) levels in human kidney cortical biopsies. Forty kidney allografts were studied using University of Wisconsin (UW) solution (n=20), Euro-Collins (EC) solution (n=12), and modified EC solution with mannitol (M;n=8). No significant differences were found between the three solutions studied with regard to ATP, ADP, or AMP changes. The mean ATP level (nmol/mg prot±SEM) at the end of preservation in the UW group was 2.7±0.3 nmol/mg, in the EC group 3.8±0.7 nmol/mg, and in the M group 2.3±0.4 nmol/mg. ATP 30 min after reperfusion in the UW, EC, and M groups was 5.7±0.8 nmol/mg, 6.4±1.0 nmol/mg, and 4.6±0.5 nmol/mg, respectively. However, an important difference appeared in the catabolic products determined. Kidneys perfused with UW solution had a significantly higher level of adenosine (2.6±0.6 nmol/mg), inosine (11.8±2.2 nmol/mg), and hypoxanthine (18.1±2.1 nmol/mg) at hypoxanthine of cold storage than those perfused with EC (0.4±0.1 nmol/mg, 2.0±0.8 nmol/mg, and 7.1±1.4 nmol/mg) and M solutions (0.2±0.05 nmol/mg, 0.5±0.1 nmol/mg, and 5.2±0.6 nmol/mg; P<0.05). These levels returned to initial values 30 min postreperfusion and there were no differences with the EC or M solution groups at that time. Thus, the adenosine present in UW solution does not appear to be useful in recovering the adenine nucleotide pool at reperfusion. Moreover, it produces a marked increase in degradation products. Our findings do not support the beneficial metabolic effect of UW solution in terms of adenine nucleotide metabolism in comparison with simpler and less expensive preservation solutions like EC.