Hepatic cell membrane damage during cold preservation sensitizes liver grafts to rewarming injury

Background/Purpose. Increasing levels of glycerol in extracellular fluid indicate cell membrane disintegration, and can be continuously monitored with microdialysis. The aim of this study was to monitor liver cell membrane damage during liver transplantation in a pig model. Methods. Thirteen donor and recipient pairs were divided into two groups; group I, with a liver ischemia time of 5h (n = 6), and group II, with 15h of ischemia (n = 7). Microdialysis samples from the liver graft were collected at 20-min intervals during donor operation, cold preservation, implantation, and reperfusion in the recipient. Glycerol concentrations were analyzed. Results. During cold preservation, a continuous increase in glycerol levels was observed. This increase correlated with the time of cold ischemia (r ² = 0.88). During implantation, an accelerated increase of glycerol was observed, with a higher acceleration in group II (P 0.005). During the first hour after reperfusion, a higher release of glycerol was observed in group II (P = 0.0005). Conclusions. Our data show a constant glycerol release during cold storage, indicating a continuous injury to the cell membranes over time. Improvement in cell membrane protection during cold preservation could lead to improvement in liver transplantation outcome and acceptance of an extended period of cold ischemia.     

Fate of hepatocyte and sinusoidal lining cell function and kinetics after extended cold preservation and transplantation of the rat liver

We investigated the chronological profile of graft damage and recovery after liver cold ischemia-reperfusion (I/R) injury, with particular attention to the role of apoptosis on hepatocyte and sinusoidal endothelial cell (SEC) damage. Male Lewis rats underwent rearterialized orthotopic liver transplantation using grafts subjected to a short (University of Wisconsin [UW] solution for 1 hour [UW1h]) and prolonged period (UW16h) of cold preservation. Experiments were performed immediately after preservation and 4 hours, 24 hours, 3 days, and 7 days after reperfusion. At each time, graft function, incidence of apoptotic cells, expression of the epitope recognized by a monoclonal antibody specific to rat SECs (SE-1), and incidence of proliferating cells were estimated. In the UW16h group, the proportion of apoptotic SECs was markedly elevated at 4 hours. The incidence of hepatocyte apoptosis was very low, although massive hepatocyte necrosis was evident at 24 hours. The incidence of proliferating hepatocytes and SECs peaked at 3 days, then returned to normal by 7 days. SE-1 expression was reduced immediately after preservation, followed by a marked reduction at 4 and 24 hours after reperfusion, and expression returned to normal by 7 days. Although SEC apoptosis was induced in the early phase of cold I/R injury, hepatocyte damage developed without the occurrence of apoptosis. Regeneration of both hepatocytes and SECs after cold I/R injury peaked at 3 days and was complete by 7 days, whereas functional recovery of these cell populations was complete 3 days after reperfusion. (Liver Transpl 2002;8:370-381.)     

匹立尼酸对大鼠肝脏冷保存损伤的保护作用

目的:观察匹立尼酸(pirinixic acid)对大鼠肝脏冷保存损伤的保护作用。方法:将60只SD大鼠随机均分为匹立尼酸预处理组和对照组,匹立尼酸预处理组分别于术前2 d,1 d和1 h尾静脉注射匹立尼酸[3 mg(/kg.d)],对照组相应给予等体积的溶剂。切取两组大鼠肝脏,放入4℃林格氏液中保存。两组分别于保存0(保存前),2,4,6,8 h时间点各取6例肝脏标本,用RT-PCR,Western blot法检测肝组织过氧化物酶体增殖物激活剂受体α(PPARα)mRNA及蛋白表达量,免疫组织化学法检测肝细胞内核因子κB(NF-κB)活性,并行肝组织病理学检查。结果:随着冷保存时间的延长,两组肝组织中PPARαmRNA和表达蛋白的量均逐渐降低,肝组织病理学评分逐渐增高,但同一时间点匹立尼酸预处理组PPARαmRNA和表达蛋白的量高于对照组,病理组织学评分低于对照组,差异均有统计学意义(均P<0.05);除对照组在8 h时间点外,两组肝细胞内NF-κB的活化水平随着冷保存时间的延长均逐渐增高,且同一时间点匹立尼酸预处理组NF-κB活化水平低于对照组(均P<0.05)。结论:匹立尼酸对大鼠肝脏冷保存损伤具有保护作用,其机制可能与其上调PPARα的表达、抑制NF-κB的活化有关。     

肝脏低温保存时肝窦内皮细胞损伤的研究

利用低温灌注技术，电镜技术和生化检测技术，对低温保存人肝脏肝窦内皮细胞的损伤进行了研究。结果显示：低温保存时不仅肝实质细胞受到损伤，而且肝窦内皮细胞发生更严重的损伤，随着保存时间延长，嘌呤核苷磷酸化酶（ＰＮＰ）逐渐升高。秀射电镜和扫描电镜显示：人肝脏低温保存后，肝窦内皮细胞可出现变性肿胀，肝窦内大泡形成，随着保存延长，肝窦内皮细胞出现变性坏死导致肝窦阻塞。肝窦内皮细胞损伤后可导致肝脏微循环系统连续     

大鼠移植小肠冷保存/再灌注后基因表达谱的变化

目的　研究冷保存 /再灌注后大鼠移植小肠基因表达谱的变化 ,以探讨移植物损伤的机制。方法　建立改良式大鼠异位节段小肠移植和正常对照模型。抽提对照组正常小肠和移植肠冷保存 /再灌注 1h后总RNA并纯化mRNA将两组等量抽提纯化的小肠mRNA逆转录合成cDNA荧光探针 ,与cDNA芯片杂交。严格洗片后扫描芯片荧光信号图像 ,分析比较两组中差异表达基因。结果　在 40 96条基因中 ,两组间存在差异表达的基因共 82条 ,新发现基因 18条 ,表达序列标识 (EST ) 3 3条 ,已报道基因 3 1条。该 3 1条差异表达基因与移植物冷保存 /再灌注损伤存在相关性。结论　移植小肠冷保存 /再灌注损伤与中性多形核白细胞和微血管内皮细胞异常黏附、移植物细胞能量和葡萄糖、蛋白质代谢障碍等因素有关     

自制KYL液对大鼠肝脏低温保存后细胞凋亡影响的研究

目的 研究自制的KYL液对大鼠肝脏低温保存后细胞凋亡的影响。方法 采用大鼠肝脏非循环离体灌注模型（noncirculated isolated perfusion of ratliver，IPRL），随机以KYL液和UW液对大鼠肝脏保存0、4、8、16、24、48h，测定灌注流出液氧自由基代谢产物（丙二醛MDA和超氧化物歧化酶SOD）的含量，检测肝细胞内钙离子浓度，检测肝细胞凋亡率和凋亡相关基因表达，观察肝脏组织形态学变化。同时设生理盐水保存阴性对照组，了解器官保存液对大鼠肝脏有无保护作用。结果 KYL液保存的大鼠肝脏肝细胞内钙离子浓度较UW液保存者低，灌注流出液MDA和SOD含量与UW液保存者相近，两者肝细胞凋亡率及凋亡基因表达情况相近，光、电镜观察两者形态学变化基本一致。两组所有指标均较生理盐水保存组好，说明两种液体对大鼠肝脏均有保护作用。结论 自制的KYL液对大鼠肝脏的保存效果在钙拮抗方面略优于UW液，在抑制细胞凋亡方面与UW液相当，而在防止细胞水肿方面较UW液稍差。     

Sinusoidal C4d deposits in liver allografts indicate an antibody-mediated response: diagnostic considerations in the evaluation of liver allografts

There is a paucity of data concerning the correlation of complement component 4d (C4d) staining in liver allografts and antibody-mediated rejection. Data about the location and character of C4d deposits in native and allograft liver tissues are inconsistent. We performed C4d immunofluorescence (IF) on 141 fresh-frozen liver allograft biopsy samples and native livers, documented the pattern of C4d IF staining, and correlated the findings with the presence of donor-specific alloantibodies (DSAs). A linear/granular sinusoidal pattern of C4d IF was noted in 18 of 28 biopsy samples obtained after transplantation from patients with positive crossmatch and detectable donor-specific alloantibody (pos-XM/DSA) findings. None of the 59 tested biopsy samples from patients with negative crossmatch and detectable donor-specific alloantibody (neg-XM/DSA) findings were C4d-positive (P < 0.001). No significant association was found between pos-XM/DSA and C4d IF staining in other nonsinusoidal liver compartments. To compare the results of sinusoidal C4d staining with IF and 2 immunohistochemistry (IHC) techniques, C4d IHC was performed on 19 liver allograft biopsy samples in which a sinusoidal pattern of C4d IF had been noted. Sinusoidal C4d IHC findings were negative for 17 of the 19 biopsy samples; 2 showed weak and focal staining, and both patients had pos-XM/DSA findings. Portal vein endothelium staining was present in only 1 IF-stained biopsy sample (pos-XM/DSA) but in 11 IHC-stained biopsy samples (2 of the 11 samples had neg-XM/DSA findings). We conclude that sinusoidal C4d deposits detected by IF in frozen tissue samples from liver allograft recipients correlate with the presence of DSAs and an antibody-mediated alloresponse. These observations are similar to findings reported for other solid organ transplants and can provide relevant information for patient management. Further validation of IHC techniques for C4d detection in liver allograft tissue is required.     

Allogenic serum improves cold preservation of osteochondral allografts

Background  

Although several types of culture medium have been used for preservation of osteochondral allografts, the viability of chondrocytes decreases with increasing storage duration. We previously showed the University of Wisconsin solution is more suitable for graft preservation than culture medium.     

Subnormothermic machine perfusion protects against rat liver preservation injury: a comparative evaluation with conventional cold storage

Hypothermic machine perfusion (MP) of the liver has been reported to improve graft function reclaiming marginal livers, such as those from non-heart-beating donors. Livers from obese donors often have fatty infiltrates and are more susceptible to hypothermic conditions. No data exist about MP at temperatures >4 degrees C. This study evaluated liver function after organ preservation by comparing MP at 20 degrees C with conventional cold storage. METHODS: For MP, rat livers were perfused for 6 hours using an oxygenated Krebs-Henseleit (KH) solution at 20 degrees C (pH 7.4). For cold storage, livers were perfused in situ and preserved with Celsior solution at 4 degrees C for 6 hours. The reperfusion period with KH (2 hours at 37 degrees C) was performed under the same conditions both among livers preserved by MP or cold storage. Hepatic enzyme release (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], and gamma-glutamyl transferase [GGT]), bile production, and ATP levels were measured during MP and reperfusion. RESULTS: At the end of reperfusion, livers preserved by MP showed significantly decreased liver damage compared with cold storage: AST, 18 +/- 4 vs. 45 +/- 6 mU/mL (P < .01); ALT, 1.5 +/- .07 vs. 6 +/- 0.5 mU/mL (P < .01); and LDH, 82 +/- 2 vs. 135 +/- 29 mU/mL (P < .05). No difference was observed between bile production between MP and cold storage. High levels of biliary GGT and LDH were found in cold preserved livers. ATP levels were higher in livers preserved with MP compared with those preserved by cold storage. CONCLUSIONS: MP at 20 degrees C resulted in a better quality of liver preservation, improving hepatocyte survival, compared with conventional cold storage. This may provide a new method for successful utilization of marginal livers, in particular fatty livers.     

Effect of cold preservation on lymphocyte adherence in the perfused rat liver

A study was designed to determine if cold preservation induces an increase in lymphocyte adherence to liver sinusoids on reperfusion. Rat livers were stored at 1 degree C in University of Wisconsin solution for 45 min, 8 hr, or 30 hr, and then reperfused for 90 min at 37 degrees C in an isolated perfused rat liver apparatus. Just prior to reperfusion, isogeneic rat lymphocytes prepared on a Ficoll-Paque gradient were added to the perfusate. In some studies lymphocytes were labeled with a fluorescent lipophilic membrane marker. There was no change in the number of circulating lymphocytes in an anhepatic circuit. When livers were present in the circuit, lymphocytes were lost from the perfusate into the liver in all studies, with the most rapid decrease occurring within 10 min of reperfusion. The length of preservation had a marked and statistically significant effect on the rate of disappearance of lymphocytes from the perfusate. Reduction by 50% of the number of lymphocytes infused did not affect the results when expressed as percent lymphocytes remaining in perfusate. To exclude the possibility that the loss of lymphocytes into the liver was due to a damaged subpopulation of lymphocytes, two livers stored 3 for 45 min were put into the circuit in sequence. The percent reduction in cells due to exposure to a second liver was not significantly different from that observed when cells were exposed only to a single liver. Histological studies showed fluorescence-labeled lymphocytes adherent in sinusoids, and the number of labeled cells was directly related to the length of preservation. Cold preservation induces an increase in lymphocyte adherence in the reperfused liver, which might be important in graft malfunction and rejection.     

Induction of specific stress response increases resistance of rat liver allografts to cold ischemia and reperfusion injury

Heme oxygenase-1 (HO-1) has been shown to increase cellular resistance against oxidative injury, but the functional significance of this is currently obscure. We investigated the protective role of HO-1, induced by tin-protoporphyrin IX (SnPP), in attenuating liver transplantation injury. Lewis rats were intraperitoneally treated with saline as control, 50 micro mol/kg of SnPP, or 2 mg/kg of cycloheximide (CHX) before SnPP injection. Gene expression of HO-1 was induced after either treatment with SnPP- or CHX + SnPP instead of saline, whereas HO-1 protein synthesis was enhanced in Kupffer-like dendritic cells of the SnPP-treated group. Following reperfusion of liver grafts preserved for 30 h, there were fewer intercellular adhesion molecule-1-positive cells in SnPP-treated livers, significantly reduced numbers of dead cells, and enhanced graft viability. The present data suggest that increased synthesis of HO-1 protein by SnPP pre-conditioning is linked to the improved liver graft viability through inhibition of inflammatory adhesion molecules.     

Molecular damage to rat liver mitochondrial H(+)-ATPase during cold preservation with UW solution.

Deterioration of rat liver mitochondrial function during cold preservation with UW solution was studied. Mitochondria were isolated from control liver (0-hr preservation), 24-hr preserved liver, and 48-hr preserved liver with UW solution at 4 degrees C. Respiration assay revealed that phosphorylation was damaged more rapidly than oxidation. Inside-out submitochondrial particles prepared from each sample by sonication in the presence of EDTA were subjected to ATPase assay. ATP hydrolyzing activity of H(+)-ATPase (ATP synthase), which plays a key role in phosphorylation in mitochondria, decreased markedly to 58% after 24-hr preservation. After 48-hr preservation, reduction to 40% of control was noted. When an intrinsic H(+)-ATPase inhibitor protein was removed from ESMP by Sephadex gel filtration, decrease of the ATPase activity was enhanced down to 49% and 29% of the control for 24-hr and 48-hr preserved liver, respectively. Molecular damage of the enzyme was confirmed by SDS-PAGE. Alpha subunit of the enzyme decreased time-dependently, and H(+)-ATPase molecules that lost alpha subunit seemed to lose their catalytic activity. Although the cause of the molecular damage of H(+)-ATPase is not clear yet, some mitochondrial protease(s) may be involved.     

Multidrug donor preconditioning prevents cold liver preservation and reperfusion injury

Purpose  

Primary graft dysfunction still represents a major challenge in liver transplantation. We herein studied in an isolated rat liver perfusion model whether a multidrug donor preconditioning (MDDP) can not only reduce but also completely prevent cold ischemia–reperfusion injury.     

Taurine attenuates cold ischemia-reoxygenation injury in rat liver

BACKGROUND: Taurine, betaine, and inositol were recently identified as osmolytes in liver cells interfering with cell volume regulation and cell function. In this study, the effect of osmolytes on cold ischemia-reoxygenation injury was investigated in rat liver. METHODS AND RESULTS: Isolated rat livers were flushed for 15 min with Krebs-Henseleit buffer (KHB), then stored for 16 hr in KHB at 4 degrees C, and thereafter reperfused with oxygenated KHB for 180 min. When taurine, betaine, and inositol (2 mmol/L, each) were added to the preperfusion and storage buffer, lactate dehydrogenase, aspartate amino transferase, and glutathione S-transferase leakage into the effluent perfusate during the reoxygenation period were less than half compared to controls without osmolytes and bile flow was higher. The effect of taurine (2 mmol/L) was similar to a mixture of all three osmolytes, indicating that taurine is the most important constituent. When livers were stored for 24 hr in University of Wisconsin solution, osmolyte addition to the storage solution also decreased lactate dehydrogenase and aspartate aminotransferase leakage during reoxygenation. Increasing liver taurine content by a 7-day taurine supplementation of drinking water attenuated reoxygenation injury in cold and warm ischemia in rat livers, whereas taurine depletion by beta-alanine feeding had the opposite effect. CONCLUSIONS: The data show that taurine protects livers from ischemia-reoxygenation. Taurine addition to perfusion and storage solutions in low millimolar concentrations or taurine supplementation of the donor may be useful to protect transplanted organs.     

Reduction in nonparenchymal cell injury and vascular endothelial dysfunction after cold preservation of the liver by gaseous oxygen

Abstract  Reintroduction of oxygen to previously anoxic tissue may result in severe cell injury (oxygen paradox) and contribute to the so-called reperfusion damage of is-chemic organs. Our study investigated the influence of simple gas eous oxygen supply during ischemia on nonparenchymal cell alterations upon reperfusion of the liver. Livers from male Wistar rats were isolated, rinsed blood-free and stored for 48 h at 4 °C in UW-preservation solution (group 1; n = 6). Gaseous oxygen was insufflated into a second group of livers (group 2; n = 6) during the storage period via the inferior caval vein at a pressure limited to 18 mmHg. To simulate the period of slow rewarming of the organ during surgical implantation in vivo, all livers were incubated at 25 °C in saline solution for 30 min prior to reperfusion. Reperfusion was carried out in vitro in a recirculating system with Krebs-Henseleit buffer. A control group was perfused immediately after harvest. The technique of aero bic storage (group 2) resulted in normal vascular perfusion charac teristics without elevation of portal venous pressure (PVP) above control values, in contrast to group 1 livers which showed a significantly elevated PVP, averaging between 1.5 and 2 times the values of the control. Hepatic efflux of NO (nmol/ml) after 10 min of reperfusion was massively increased in group 1, while only low concentrations were found in group 2 and in control livers. Kupffer cell activation after ischemia was shown by a huge increase in acid phosphate release upon reperfusion compared with the control, with significantly lower values in group 2 after 10 min of reperfusion than in group 1. Thus, aerobic ischemia by gaseous oxygen persuf-flation seems an appropriate tool for long-term organ preservation, pre venting vascular and parenchymal dysfunction upon reperfusion.     

The mechanism of injury in a steatotic liver graft during cold preservation

BACKGROUND: Fatty livers are more prone to primary nonfunction after transplantation. It is known that cell injury is strongly associated with alterations in the content and composition of membrane lipids. We assumed that plasma membrane (PM) fluidity, which is the most important property of the membrane, differed between fatty and normal livers. METHODS: The livers from obese and lean Zucker rats were flushed with cold Ringer's lactate and University of Wisconsin (UW) solution via the portal vein and preserved in cold UW solution for 24 hr. Histological examinations of electron microscopy were performed to investigate of sinusoidal lining cells (SLCs). PMs were isolated using a discontinuous density gradient of Percoll, and the lipid compositions were determined by chromatography. RESULTS: SLCs of fatty livers were markedly injured compared with control livers even after short preservation time. Moreover, many blebs were observed in the obese rats even after short preservation time. As for PM lipid composition, the cholesterol/phospholipid (PL) ratio of total PM was 0.14+/-0.03 in the obese rats and 0.21+/-0.03 in the lean rats (P<0.05). The relative proportions of polyunsaturated fatty acids among PLs in PM were 35.7+/-1.2% vs. 45.9+/-1.5% (P<0.0001). These results indicated that the fluidity of the PM in the obese rats is decreased after exposure to low temperatures. CONCLUSIONS: Our results suggest that steatotic livers from obese donors are more susceptible to cold preservation injury than livers without steatosis because of the severe deterioration of SLCs, and it is associated with PM fluidity even after short-term cold preservation.     

Cyclosporine overcomes cold preservation/reperfusion injury of liver graft: Chemokine release and liver ultrastructure

There is a growing body of evidence that the cytokine, tumor necrosis factor-α (TNF-ga), plays an important role in the development of hepatic ischemia/reperfusion injury. We found that the immunosuppressants, cyclosporine-A (CsA), azathioprine, and FK506, have protective effects on such injury. The purpose of the present study was to elucidate mechanisms involved in these beneficial effects of the immunosuppressant, CsA, on liver injury following cold preservation and transplantation, with special reference to the suppression of TNF-α release. Rat livers were stored in Euro-Collins solution (EC) at 4°C for 6h and orthotopically transplanted. The animals allotted to two groups: group A (untreated controls) and group B (CsA pretreatment of recipients). CsA (10 mg/kg, p.o.) was given for 3 consecutive days preoperatively. CsA pretreatment of the recipients significantly improved the 2-week survival rate (0/6 for group A, 3/6 for group B;P<0.05) and this was associated with a significant decrease in serum TNF-α levels 2h posttransplantation (group A, 69.8±15.7 pg/ml; group B, 22.8±6.8; mean±SEM;n=12 each;P<0.05) and amelioration of sinusoidal endothelial injury, assessed by electron microscopy. Plasma endotoxin levels following reperfusion of the grafts were not altered by the CsA therapy. Morphologically, CsA pretreatment of the recipients did not alter activation of Kupffer cells. CsA pretreatment of the recipient aids in preventing cold preservation/reperfusion injury of the liver graft, possibly by modulating effects of TNF-α.     

大鼠供肝冷缺血时间与肝移植术后受者肺损伤的关系

目的 探讨大鼠供肝冷保存时间与肝移植术后受者肺损伤的关系。方法 用Wismr大鼠建立原位肝移植模型，按供肝冷保存时间的不同分为5组（n=10），A组为假手术组，B组、C组、D组及E组的供肝冷保存时间分别为45min、90min、120min及180min。各组大鼠在移植肝恢复血流180min后处死，观察肺组织病理学变化，测定肺组织湿干重比值（W／D）、肺组织中髓过氧化氢酶（MPO）的活性，同时检测肝脏流出道血清肿瘤坏死因子（TNF-α）和白细胞介素18（IL-1β）的水平以及肺组织中TNF-α和IL-1β的表达。结果随着供肝冷保存时间的延长，肝脏流出道血清TNF-α和IL-1β水平及肺组织中TNF-α和IL-1β的表达均逐渐升高，肺组织W／D和MPO活性逐渐增高，肺损伤加重。结论供肝冷缺血时间与肝移植术后受者肺损伤相关，肺损伤随供肝冷缺血时间的延长而加重；血清中TNF-α和IL-1β升高及肺组织中TNF-α和IL-1β表达上调可能与肝移植后的肺损伤有关。