Preservation injury of jejunal grafts and its modulation by custodiol and university of wisconsin perfusion solutions in wistar rats

BACKGROUND: The two preservation solutions most commonly used in human transplantation surgery are University of Wisconsin (UW) and Custodiol (histidine-tryptophan-ketoglutarate; HTK). The aim of our study was to compare the protective effect of UW and HTK solutions on preservation-induced injury of jejunal grafts, as evaluated by the histological changes (semiquantitative method) and small bowel mucosal serotonin levels (as a possible new quantitative method). METHODS: Male Wistar rats (n = 50) weighing 316 +/- 52 g were divided into two main groups according to which preservation solution was used, i.e. UW (n = 25) or HTK (n = 25), and each of these groups was divided into five subgroups according to cold ischemic time (0, 1, 6, 9 and 12 h). Jejunal mucosa biopsy specimens were obtained to determine the serotonin concentration in mucosa and for standard light histology. To grade histological changes in mucosa, Park's small bowel injury grading system was used. RESULTS: Histological examination revealed that injury increased with cold ischemic time in the UW as well as in the HTK group, and there were no significant differences in injury between the two groups, except for the 6-hour cold ischemic period (p < 0.05), when HTK-preserved grafts showed a lower degree of injury (0.97 +/- 0.41) compared with UW-preserved grafts (1.25 +/- 0.39). The mucosal serotonin concentration decreased with cold ischemic time in both groups, and there were significant differences (p < 0.05) in concentrations between the groups after 9 and 12 h of cold ischemia. A significantly higher concentration was measured in grafts preserved in UW solution at these time points. CONCLUSION: The concentration of mucosal serotonin in rat small bowel grafts preserved for 9 and 12 h in UW preservation solution was significantly higher than that in HTK solution. These findings indicate a better protective effect of UW solution on small bowel injury after 9 h of cold ischemia.     

Heat shock inhibits NF-kB activation in a dose- and time-dependent manner

BACKGROUND: The heat shock response (HSR) attenuates NF-kappaB mediated activation of the acute inflammatory response by inhibiting IkB degradation. The HSR also confers a protective phenotype upon cells through production of heat shock proteins (HSP). However, the exact conditions that induce the HSR and stimulate the production of protective HSP are poorly defined. Consequently, we hypothesized that the inhibition of NF-kappaB activation through the HSR is dependent both on the degree of cellular injury and the length of the recovery period from the heat shock. METHODS: RAW 264.7 murine macrophages were heated to 43 degrees C for 15 (mild heat shock), 45 (moderate heat shock), or 90 min (severe heat shock), allowed to recover at 37 degrees C for 0 to 24 h, and then exposed to 100 ng/ml of Escherichia coli (055:B5) lipopolysaccharide (LPS). Cellular viability, HSP expression, and the activation of NF-kappaB after LPS exposure were determined by alamarBlue assay, immunoblot, and electrophoretic mobility shift assay, respectively. RESULTS: Transient attenuation of NF-kappaB activation and IkappaB preservation was observed only with moderate heat shock and 1 h of recovery. Mild heat shock had no effect on LPS-induced NF-kappaB activation or IkappaB degradation. Severe heat shock completely inhibited NF-kappaB activation and preserved IkappaB protein levels. Heat shock proteins were detectable 30 min after moderate heat shock, with maximal and sustained levels 2 to 24 h after heat shock. CONCLUSION: The attenuation of NF-kappaB activation after heat shock is both dose- and time-dependent.     

Comparison of solutions for preservation of the rabbit liver as tested by isolated perfusion

The University of Wisconsin (UW) solution consists of a relatively complex mixture of agents. In this study we compared simpler preservation solutions, namely, histidine-tryptophan-ketoglutarate glutarate (HTK) and phosphatebuffered sucrose (PBS) with different compositions of UW solution in the isolated perfused rabbit liver model. Livers were stored cold for 24 and 48 h. After 24 h of preservation, the amount of bile produced in UW-preserved livers was significantly greater (P<0.05) than that in HTK-preserved livers. Also, there was less LDH released into the perfusate in UW-preserved livers. There was more edema and lower K+/Na+ rations in HTK-preserved livers than in UW-preserved livers (all data P<0.05). After 48 h of preservation, the differences between livers preserved in UW or HTK solution were less noticeable than at 24 h and bile production was similar. LDH and AST release were greater in HTK-preserved livers than in UW livers, but these differences were not statistically significant. Preservation in PBS for 48 h was worse than in either UW or HTK solution. Substitution of polyethylene glycol (PEG) for hydroxyethyl starch (HES) in 48-h UW-preserved livers was not effective. We conclude that solutions simpler in composition than UW solution may be effective in kidney transplantation but do not appear suitable for successuful liver preservation.     

A comparison of Collins and UW solutions for cold ischemic preservation of the rat liver

A new solution which can extend successful preservation times for hepatic allografts was recently developed at the University of Wisconsin (UW). To examine the mechanism of improved viability using this solution, we developed a model of orthotopic hepatic transplantation in the rat. As a baseline study, we compared parameters of viability of allografts preserved in Collins solution to those preserved in UW, including survival, bile output, peak AST, and allograft weight change during storage. Seventy-four rats were transplanted following storage in Collins solution and 70 rats were transplanted after storage in UW. Cold-storage time varied between 2 and 24 hr. The survival with preservation in UW was significantly better than that with Collins when storage time was greater than 2 hr. The preservation time for a viable organ using UW was greater than double that using Collins. The peak AST using UW was lower than that with Collins for cold ischemic times (CIT) up to 10 hr, with significance demonstrated at 5-6 and 7-8 hr when compared with Collins. Prolonged CIT resulted in an increase in liver weight with Collins-preserved livers and a decrease in weight with UW-preserved livers. Using a model of orthotopic liver transplantation in the rat, we demonstrated a doubling of preservation time when UW solution was substituted for Collins. Similar improvements in recipient survival and biochemical parameters of injury have been demonstrated in the canine model and in human clinical trials.     

Heme oxygenase-1 attenuates ischemia/reperfusion-induced apoptosis and improves survival in rat renal allografts

BACKGROUND: Kidneys can be preserved only for a limited time without jeopardizing graft function and survival. Induction of heat shock proteins (HSPs) can protect against ischemia/reperfusion (I/R) injury. Therefore, we investigated whether the induction of the HSP, heme oxygenase-1 (HO-1), improves outcome following isotransplantation after an extended period of cold storage. METHODS: Rats were subjected to heat preconditioning (HP; 42 degrees C for 20 minutes). Kidneys harvested after 24 hours, were preserved in cold University of Wisconsin (UW) solution at 4 degrees C for 45 hours and transplanted into bilateral nephrectomized rats. Cobalt protoporphyrin (CoPP) was administered in another group of animals in order to induce HO-1 pharmacologically, while other groups of animals received the HO-1 inhibitor, tin protophorphyrine (SnPP), following HP or CoPP. RESULTS: Cold ischemia caused a complete attenuation of graft function within 3 days following transplantation and subsequent death of all animals, whereas HP protected graft function and five of nine rats survived for 3 weeks. HP inhibited the induction of osteopontin and induced the expression of HO-1, HSP 70 and 90, and the antiapoptotic factor Bcl-XL. Grafts exposed to HP were protected against structural I/R injuries as revealed by histologic assessment using a semiquantitative score. Furthermore, induction of apoptosis was attenuated and activation of caspase-3 was inhibited. Comparable results were observed after administration of CoPP, whereas SnPP inhibited the effects of HP and CoPP. CONCLUSION: HP or administration of CoPP induced both HO-1, preserved kidney graft function, and prevented postreperfusion apoptosis after cold preservation.     

阿霉素诱导热休克蛋白72对大鼠肝脏冷缺血-再灌注损伤的保护作用

目的观察阿霉素（DXR）预处理诱导大鼠肝脏热休克反应在肝脏长时间冷缺血一再灌注损伤中对肝细胞的保护作用。方法供体大鼠术前按1mg／kg由外周静脉注射DXR（DXR组）,对照组注射生理盐水。48h后行肝脏原位冷灌注,获取肝脏后将其在4℃UW液中保存48h,然后行原位肝移植,再灌注1、3h。逆转录-聚合酶链反应法测定肝组织肿瘤坏死因子-α（TNF-α）mRNA、中性粒细胞化学趋化性细胞因子（CINC）mRNA、巨噬细胞炎症蛋白-2（MIP-2）mRNA的表达,蛋白质印迹法测定肝组织热休克蛋白72（HSP72）、核转录因子-κB（NF-κB）的表达,测定血清谷丙转氨酶、TNF-α、CINC、MIP-2水平。同时观察7d生存率。结果DXR组TNF-α mRNA、CINCmRNA、MIP-2mRNA的表达均低于对照组。DXR组HSP72表达显著,对照组基本无表达;DXR组NF-κB无表达,对照组显著表达。DXR组血清TNF-α、CINC、MIP-2显著低于对照组（P〈0．05）。DXR组7d生存率为50％,对照组为0（P〈0．05）。结论DXR预处理大鼠供肝可使肝脏长时间冷缺血-再灌注损伤显著减轻;HSP72的诱导可抑制NF-κB激活导致的炎症反应,对肝实质细胞提供保护作用。     

Protective effect of heat preconditioning of rat liver graft resulting in improved transplant survival

BACKGROUND: The protective effect of heat preconditioning of the liver against ischemia-reperfusion injury has been reported mostly in models of transient ischemia in relation to heat shock protein 70 (HSP70). We estimated the effect of heat preconditioning of liver grafts on the transplant survival rate and on apoptosis of sinusoidal endothelial cells (SEC) as well as hepatocytes in a rat model of liver transplantation. METHODS: Donor rats of the heat shock (HS) group were subjected to heat preconditioning 48 hr before graft harvest, and HSP70 levels were estimated by. Western blot analysis and immunohistochemistry. The liver isografts from the HS group and control (C) group were preserved in Euro-Collins solution for 6 or 8 hr and transplanted orthotopically. Serum hyaluronic acid and alanine aminotransferase were measured, and apoptosis of the SEC and hepatocytes was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining and electron microscopy. RESULTS: HSP70 expression was detected not only in hepatocytes but also in SEC. In the 8-hr preservation model, the 1-week survival rate was 60% in the HS group and 0% in the C group. Serum hyaluronic acid and alanine aminotransferase levels in the HS group were significantly lower than those in the C group at 3 hr after reperfusion, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling-positive SEC in the C group (35.2%) was markedly increased compared with the HS group (10.1%). Electron microscopic examination confirmed the features of apoptosis of SEC. CONCLUSIONS: Heat preconditioning of the graft improved the survival rate of the liver transplants. Induction of HSP70 in SEC as well as in hepatocytes might attenuate preservation-reperfusion injury by inhibiting apoptosis of SEC.     

Heat preconditioning attenuates renal injury in ischemic ARF in rats: role of heat-shock protein 70 on NF-kappaB-mediated inflammation and on tubular cell injury

Although heat preconditioning has been known to be protective in various types of injury, the precise molecular mechanism for this is unclear. Recent observations that indicate that previous heat shock has an anti-inflammatory, antiapoptotic effect led to this investigation of the in vivo effect of heat preconditioning on NF-kappaB activation and inflammation and also on tubular cell injury in ischemic acute renal failure (ARF). Heat preconditioning provided marked functional protection and also reduced histologic evidence of tubular necrosis. Ischemia/reperfusion-induced NF-kappaB activation was suppressed by heat preconditioning with a subsequent decrease in monocyte chemoattractant protein-1 expression and inflammatory cell infiltration. Heat preconditioning also suppressed the accumulation of phosphorylated inhibitory kappaBalpha (IkappaBalpha) with a resultant depletion of cytoplasmic IkappaBalpha, indicating that heat preconditioning blocked the activation of the IkappaB kinase complex. Tubular cell apoptosis, determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, also was decreased by heat preconditioning, and this was accompanied by decreased caspase 3 activation. Among several heat-shock proteins (HSP), HSP-70 was induced primarily by heat preconditioning. Inhibition of HSP-70 by quercetin almost completely reversed the functional protection that was provided by heat preconditioning. These data provide evidence that HSP-70 affords protection via inhibition of NF-kappaB-mediated inflammation and also inhibition of the cell death pathway in ischemic ARF. Further elucidation of the cytoprotective mechanism of stress proteins could facilitate new target or drug development in the treatment of ARF.     

Genetic modification of cold-preserved renal grafts using HSP70 or bcl-2 HVJ-liposome method

BACKGROUND: We tested the hypothesis that the best time for genetic modification is while the cell viability of the graft is reduced for long-term preservation. The hemagglutinating virus of Japan (HVJ)-liposome method, a nonviral gene transfer technique, was used with a luciferase gene to test the efficacy of protein induction under the critical preservation time. Furthermore, we tested this genetic modification with heat shock protein (HSP) 70 or bcl-2 genes to prevent primary nonfunction (PNF) after long-term preservation. METHODS: Orthotopic rat renal transplantation (RT) was performed using the cuff technique in the syngeneic combination of Lew (major histocompatible complex, haplotype: RT1(l)). Rat kidney grafts were preserved for 24 or 48 h in University of Wisconsin (UW) or Ringer's lactate solution using HVJ method with the luciferase gene. Rats with gene-transfected kidneys were re-laparotomized 48 h after transplantation to estimate the lack of arterial flow in the graft and killed for histological evaluation of the degree of PNF luciferase intensity assay. Then, two functional genes (HSP70 or bcl-2) were tested for the occurrence of PNF and histological and immunohistochemical analysis of the grafted kidneys preserved for 48 h in the UW solution. RESULTS: In the kidneys preserved for 24 h, 50% of the Ringer's lactate group had PNF; but all of the UW group had sufficient blood flow. The graft viability was well corrected by the degree of luciferase intensity. The PNF rate was significantly suppressed in the bcl-2 gene-transfer group, and tended to be reduced in the HSP70 group. CONCLUSIONS: The HVJ-liposome method effectively induced the foreign gene for kidney grafts even in the cold-preservation solution. Induction of bcl-2 or the HSP70 gene reduced the occurrence of PNF in the rat renal graft. The results suggest that gene transfer not only maintains graft viability, but also graft activation.     

Intraoperative evaluation of EuroCollins and University of Wisconsin preservation solutions in patients undergoing hepatic transplantation

The effects of cold liver preservation with two solutions, EuroCollins and University of Wisconsin, were compared in terms of hepatic function and hemodynamic parameters obtained intraoperatively during orthotopic liver transplantation. Data from 101 consecutive liver transplants were analyzed retrospectively, comparing 50 grafts preserved with EC with 51 preserved with UW solution. Hepatic hemodynamics parameters included portal venous and hepatic arterial flows, determined with an electromagnetic flowmeter. Vascular pressures, blood gases and pH measurements were obtained directly from the portal vein, hepatic vein, and peripheral artery. Serial measurements of serum glucose, SGOT, and SGPT were performed following reperfusion. Preservation related graft failure occurred in 4 of 50 patients in the EC group, but not in any of 51 patients in the UW group. Cold Ischemia time (hours +/- SEM) was significantly prolonged in UW group (7.23 +/- 1.4 vs. 5.21 +/- 0.9). Rate of temperature change (degrees C/hour +/- SEM) after reperfusion was similar in both groups (EC = 0.62 +/- 0.35, UW = 0.71 +/- 0.4). Peak serum SGOT, SGPT, and glucose levels following reperfusion were significantly higher in the EC group, as was PRBC and FFP administration. Systemic hemodynamics in both groups of patients were similar. However, UW-preserved grafts demonstrated a significantly higher hepatic artery resistance, with no other differences in hepatic hemodynamics seen. UW solution appears to extend cold ischemia time without adversely affecting liver function. However, the etiology and clinical significance of the increased hepatic artery resistance seen in UW-stored liver grafts are unknown.     

Does University of Wisconsin solution harm the transplanted heart?

BACKGROUND: University of Wisconsin solution (UW) has been shown to be an effective preservative for the cardiac allograft. Recently, the high potassium content of UW has been implicated in causing coronary endothelial damage, allegedly contributing to development of cardiac allograft vasculopathy (CAV) and eventually to poorer survival. METHODS: We examined our experience using UW for preservation of cardiac allografts between 1990 and 1994 (n = 94), and compared these to hearts preserved with the lower potassium-containing Stanford solution used at our center between 1986 and 1990 (n = 65). Indices of graft function, ischemic injury, CAV incidence, CAV severity, and survival were evaluated. RESULTS: The 2 groups were similar in age, gender, diagnosis, donor inotropic support, donor-recipient weight ratio, incidence of acute graft failure, and cytomegalovirus seroconversion. UW-preserved hearts came from older donors (30.5 vs 24.1 years, p < .001), and were transplanted into more status 1 recipients (56% vs 22%, p < .001), consistent with current trends. Mean ischemic time of UW-preserved hearts was significantly longer (184 vs 155 minutes, p < .005) although time required to wean from bypass was less (45.5 vs 73.8 minutes, p < .001) and there was a trend towards less inotropic requirement. CPK-MB release was less with UW preservation (63 vs 87 microg/ dL, p = .001). Three years after transplantation, both groups were similar in the incidence of CAV (UW, 27.3%; STNF, 37.5%; p = 0.27), and also the severity of CAV (p = 0.78). Deaths attributed to CAV were equal in each group (UW, 11.4% vs STNF, 10.7%; p = 0.79). Kaplan-Meier survival analysis revealed equivalent survival curves (p = 0.26). CONCLUSIONS: We conclude that UW is a safe and effective myocardial preservative, allowing longer ischemic times with equivalent graft function. Our data suggest that when UW is used for cardiac allograft preservation, both CAV and survival are comparable to the experience with other preservatives containing lower concentrations of potassium.     

Cytochrome P-450 content and activity after cold storage of rat hepatocytes in university of wisconsin and sodium-lactobionate-sucrose solutions.

We compared the capacity of University of Wisconsin (UW) and of sodium-lactobionate-sucrose (SLS) hypothermic preservation solutions to maintain the integrity of the hepatic cytochrome P-450-dependent mono-oxygenase system. Isolated rat hepatocytes were stored for 0, 10, 24, and 48 hours in UW or SLS solution and were subsequently cultured shortly at 37 degrees C. Cell viability declined slightly but significantly in a time-dependent manner during cold preservation in either UW or SLS solution, and warm culture exacerbated this effect. Total cytochrome P-450 declined gradually after cold preservation and warm culture to reach values of 70% and 52% of unstored controls in cells preserved for 24 and 48 hours in cold UW solution, respectively. Storage in cold SLS solution yielded a similar decrease to 79% and 59% of unstored controls for the equivalent preservation times. Cytochrome P-450 activity was assessed by the metabolism of theophylline after various cold preservation times in UW or SLS solutions. Production of the major metabolite 1,3-dimethyluric acid was not significantly affected by extended cold preservation periods in either UW or SLS solutions. Similarly, the amount of residual theophylline remained stable in all groups, suggesting that alternative metabolic routes were not modified. These studies show that cold preservation in SLS solution is as effective as that in UW solution in terms of cell viability, cytochrome P-450 content, and activity toward theophylline. In addition, the significant reduction in cytochrome P-450 in conjunction with unaffected theophylline disposition suggests that certain cytochrome P-450 isoforms are specifically damaged by cold preservation and rewarming.     

Advantages of normothermic perfusion over cold storage in liver preservation

BACKGROUND: To minimize the ischemia-reperfusion injury that occurs to the liver with the current method of preservation and transplantation, we have used an extracorporeal circuit to preserve the liver with normothermic, oxygenated, sanguineous perfusion. In this study, we directly compared preservation by the standard method of simple cold storage in University of Wisconsin (UW) solution with preservation by perfusion. METHODS: Porcine livers were harvested from large white sows weighing between 30 and 50 kg by the standard procedure for human retrieval. The livers were preserved for 24 hr by either cold storage in UW solution (n=5) or by perfusion with oxygenated autologous blood at body temperature (n=5). The extracorporeal circuit used included a centrifugal pump, heat exchanger, and oxygenator. Both groups were then tested on the circuit for a 24 hr reperfusion phase, analyzing synthetic function, metabolic capacity, hemodynamics, markers of hepatocyte and reperfusion injury, and histology. RESULTS: Livers preserved with normothermic perfusion were significantly superior (P=0.05) to cold-stored livers in terms of bile production, factor V production, glucose metabolism, and galactose clearance. Cold-stored livers showed significantly higher levels of hepatocellular enzymes in the perfusate and were found to have significantly more damage by a blinded histological scoring system. CONCLUSIONS: Normothermic sanguineous oxygenated perfusion is a superior method of preservation compared with simple cold storage in UW solution. In addition, perfusion allows the possibility to assess viability of the graft before transplantation.     

Evaluation of preservation solutions by ESR-spectroscopy: superior effects of University of Wisconsin over Histidine-Tryptophan-Ketoglutarate in reducing renal reactive oxygen species

Despite the causative role of oxidative stress in renal ischemia-reperfusion (I-R) injury effects of preservation solutions on reactive oxygen species (ROS) release have not been sufficiently evaluated. We compared the effects of most common solutions in kidney transplantation, University of Wisconsin (UW) and Histidine-Tryptophan-Ketoglutarate (HTK). ROS formation in isolated perfused rat kidney was detected by electron spin resonance spectroscopy using spin label 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Donor kidneys from Lewis rats were pretreated with saline (controls), in therapeutic groups, kidneys underwent 18 h of cold storage (CS) preserved by HTK or UW solution. Experimental protocol included a stabilization period followed by additional I-R. Kidneys preserved by HTK produced highest ROS values in the control period after CS, whereas levels in UW and control group did not vary significantly. A peak release induced by additional I-R was also significantly highest in HTK kidneys, and UW did not differ from controls. During reperfusion, levels in HTK exceeded control and UW values. Renal vascular resistance, caspase-3-activity, and tissue hydration were enhanced in HTK compared with UW group, whereas ATP concentration was less reduced in UW-preserved tissue. These data show the greater antioxidative potential of UW solution, which also attenuated organ impairment after CS in the early reperfusion period.     

Effect of S-nitrosoglutathione (GSNO) added to the University of Wisconsin solution (UW): I) Morphological alteration during cold preservation/reperfusion of rat liver

Cold liver preservation in the University of Wisconsin solution (UW) followed by reperfusion alters hepatic parenchyma and stroma. In this study we demonstrated the benefit of adding S-nitrosoglutathione (GSNO) to the UW solution before cold storage, as an effective Nitric Oxide (NO) donor to prevent hepatic injury. Wistar adult rat livers were stored in UW solution (4 degrees C-48Hs) and then reperfused 60 minutes in the isolated perfused rat liver model (IPRL). Normal untreated livers and perfused livers, but not preserved were used as controls. Parenchymal damages were evaluated with Hematoxylin-Eosin stain and an inmunohistochemistry assay for albumin was used as functional test. To study the stroma, collagen type III and I networks were analyzed using Picro-sirius Red stain and Gordon Sweets' method for reticulin. After 48 Hs of cold preservation in UW solution livers showed few rounded endothelial cells inside sinusoidal lumen and extended areas of cell vacuolation. Albumin distribution was evident only around central veins and middle zones of the hepatic lobule. Collagens III and I networks were disorganized. When preserved with the addition of 100 microM GSNO and then reperfused, the hepatic morphology, in general, was conserved showing little vacuolation, fewer endothelial cells inside sinusoids and good albumin distribution around central veins and middle zones. The stroma had organized networks of collagen III and I. We concluded that the addition of 100 microM GSNO as a NO donor, can improve UW solution properties to preserve rat liver by maintaining the hepatic morphology and avoiding hepatic injury post cold preservation/reperfusion.     

Induction of necrosis and DNA fragmentation during hypothermic preservation of hepatocytes in UW,HTK, and Celsior solutions

Donor cells can be preserved in University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), or Celsior solution. However, differences in efficacy and mode of action in preventing hypothermia-induced cell injury have not been unequivocally clarified. Therefore, we investigated and compared necrotic and apoptotic cell death of freshly isolated primary porcine hepatocytes after hypothermic preservation in UW, HTK, and Celsior solutions and subsequent normothermic culturing. Hepatocytes were isolated from porcine livers, divided in fractions, and hypothermically (4 degrees C) stored in phosphate-buffered saline (PBS), UW, HTK, or Celsior solution. Cell necrosis and apoptosis were assessed after 24- and 48-h hypothermic storage and after 24-h normothermic culturing following the hypothermic preservation periods. Necrosis was assessed by trypan blue exclusion, lactate dehydrogenase (LDH) release, and mitochondrial 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. Apoptosis was assessed by the induction of histone-associated DNA fragments and cellular caspase-3 activity. Trypan blue exclusion, LDH release, and MTT reduction of hypothermically preserved hepatocytes showed a decrease in cell viability of more than 50% during the first 24 h of hypothermic preservation. Cell viability was further decreased after 48-h preservation. DNA fragmentation was slightly enhanced in hepatocytes after preservation in all solutions, but caspase-3 activity was not significantly increased in these cells. Normothermic culturing of hypothermically preserved cells further decreased cell viability as assessed by LDH release and MTT reduction. Normothermic culturing of hypothermically preserved hepatocytes induced DNA fragmentation, but caspase-3 activity was not hanced in these cells. Trypan blue exclusion, LDH leakage, and MTT reduction demonstrated the highest cell viability after storage in Celsior, and DNA fragmentation was the lowest in cells that had been stored in PBS and UW solutions. None of the preservation solutions tested in this study was capable of adequately preventing cell death of isolated porcine hepatocytes after 24-h hypothermic preservation and subsequent 24-h normothermic culturing. Culturing of isolated and hypothermically preserved hepatocytes induces DNA fragmentation, but does not lead to caspase-3 activation. With respect to necrosis and DNA fragmentation of hypothermically preserved cells, UW and Celsior were superior to PBS and HTK solutions in this model of isolated porcine hepatocyte preservation.     

Pancreas preservation with University of Wisconsin and Celsior solutions

BACKGROUND: Although the use of Celsior has been recently described for heart, lung, liver, and kidney transplantation, no data are available on its use for clinical pancreas preservation. METHODS: We herein describe the results of 112 pancreas transplants preserved with either University of Wisconsin (UW; (n = 56) or Celsior (n = 56) solution at two Italian transplant centers. The groups were comparable with regard to all donor and recipient characteristics. RESULTS: Mean cold and warm ischemia times were 10.1 +/- 2.2 hours and 37.2 +/- 8.2 minutes for UW compared to 10.8 +/- 2.4 hours and 38.3 +/- 6.7 minutes for Celsior (P = NS). Delayed endocrine pancreas function was recorded in two UW-preserved grafts (3.6%). Actuarial 1-year patient survival was 94.6% for UW as compared with 100% for Celsior (P = NS). Equivalent graft survival figures were 91.0% for UW as compared with 96.4% for Celsior (P = NS). CONCLUSIONS: Within the range of cold ischemia times reported in this study, UW and Celsior solutions have similar safety profiles for pancreas transplantation.     

Improved preservation of the rat liver for orthotopic liver transplantation: use of University of Wisconsin-lactobionate solution and retrograde reflushing

This study investigated cold preservation and reflushing before orthotopic liver transplantation by examining (1) new University of Wisconsin solution (UW) versus Euro-Collins solution (EC), (2) retrograde reflushing (RR) versus antegrade reflushing (AR), and (3) the addition of a platelet-activating inhibitor (PAF), superoxide disumatase (SOD), or SOD + catalase to UW. Syngeneic, male Lewis rats (200 to 400 gm) were used. Preservation for 9, 12, 18, or 24 hours in UW or EC with RR (through the inferior vena cava) was used. The 9- and 12-hour groups experienced a significant decrease in the weight of the grafts preserved in UW. The 3-week survival rate after 9 hours of preservation (n = 6) in UW was 66%, and the survival rate with EC was 0% (p less than 0.025). After 12 hours of preservation, recipient survival rate was 70% (n = 10) with UW versus 0% (n = 4) with EC (p less than 0.025). RR of the graft with cold lactated Ringer's solution immediately before reimplantation significantly improved 3-week survival in the 12-hour group to the level of the control group (no preservation time, 69%). Preservation for 12 hours in UW followed by AR yielded a 3-week survival of 14%; 3-week survival for the RR group was 70% (p less than 0.025). Furthermore, RR allowed a 3-week survival of 33% and 20% after 18 and 24 hours of UW preservation, respectively. In the 24-hour RR/UW group, donor pretreatment with SRI 63-441 (20 mg/kg, intravenously) and recipient treatment with SOD (15 mg/kg, intravenously) or SOD + catalase (15 mg/kg and 5000 units/kg, intravenously) produced a 3-week survival comparable to preservation in UW followed by RR alone. These studies show that UW is a profound improvement over EC for cold preservation of liver and that the new application of RR to rat orthotopic liver transplantation improves survival. However, the addition of free-radical scavengers or PAF does not improve organ function or recipient survival in this model.     

Heat shock response inhibits NF-kappaB activation and cytokine production in murine Kupffer cells

BACKGROUND: Kupffer cells play a crucial role in the pathogenesis of sepsis through production of proinflammatory mediators and control of systemic endotoxemia. The anti-inflammatory effects of heat shock response (HSP) have been well documented. However, the role of HSP in lipopolysaccharide (LPS) induced Kupffer cell activation has not been fully investigated. In this study, we investigated the effects of HSP on LPS induced Kupffer cell NF-kappaB activation and cytokine production. MATERIALS AND METHODS: Kupffer cells were isolated from mice by collagenase digestion and HSP was induced by culturing Kupffer cells with sodium arsenite. Kupffer cells were stimulated in vitro by LPS. Heat shock protein (HSP)-70 expression and cytoplasmic IkappaBalpha protein was determined by Western blot. Supernatant tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-10 levels were measured by ELISA. NF-kappaB activation was analyzed by electrophoresis mobility shift assay. Cytokine and IkappaBalpha mRNA expression were determined by RT-PCR. Toll-like receptor 4 expression on Kupffer cells was determined by flow cytometry. RESULTS: HSP pre-conditioning significantly inhibited LPS-induced cytokine TNF-alpha and IL-6 production and mRNA expression. NF-kappaB activation and IkappaBalpha degradation induced by LPS were attenuated by HSP. HSP up-regulated expression of IkappaBalpha mRNA. No effect of HSP on cell surface expression of TLR4 was observed. CONCLUSIONS: Increased IkappaBalpha stability and up-regulation of IkappaBalpha gene expression may be one of the mechanisms of the inhibition of LPS induced Kupffer cell activation by HSP. HSP also inhibited expression of the anti-inflammatory cytokine IL-10, and the mechanism and biological significance of this effect merit further investigation.     

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.)