University Of Wisconsin Storage Research Articles

Advantages of normothermic perfusion over cold storage in liver preservation.

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. 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. 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. 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.

Respiratory Activity of Isolated Rat Hepatocytes Following Cold Storage and Subsequent Rewarming: A Comparison of Sucrose-Based and University of Wisconsin Solutions

Investigations were carried out on the respiratory function of isolated rat hepatocytes after cold storage alone for periods up to 48 h in either sucrose-based solution (SBS) or University of Wisconsin (UW) solution and after subsequent normothermic preincubation. In both SBS and UW, cold storage for 24 h depressed respiratory function (to 21 ± 3 and 23 ± 3 nmol O2/min/106 cells, respectively) compared to control cell values (31 ± 3 and 33 ± 5 nmol O2/min/106 cells; P < 0.01 in each case). However, normothermic preincubation for 60 min returned respiratory activity to control values (for SBS and UW storage: 41 ± 6 and 40 ± 5 nmol O2/min/106 cells; for control cells: 43 ± 5 and 46 ± 6 nmol O2/min/106 cells). Storage for 48 h in both SBS and UW allowed further depression of respiratory activity, with no recovery after preincubation. Stimulation of respiration by succinate in hepatocytes stored for longer periods was suggestive of increased membrane permeability. Both SBS and UW are effective storage solutions for isolated hepatocytes for up to 24 h as judged by aerobic metabolism, but significant damage was expressed in both solutions when preservation was extended.

Seventy-two hours hypothermic intestinal preservation study using a new perfluorocarbon emulsion.

We investigated the effect of a perfluorocarbon emulsion (FC) added to the University of Wisconsin (UW) solution on hypothermic (4 degrees C, 12-72h) preservation of rat small bowel grafts. The FC was 90%w/v perfluorooctylbromide, 2%w/v egg yolk phospholipids and 1.4%w/v mixed fluorocarbon-hydrocarbon molecular dowels. Four groups were defined: [1] UW flush and UW storage; [2] UW flush and FC storage; [3] flush with FC diluted 2 times with UW (FU) and FU storage; [4] FU flush and storage in oxygenated FU. Preservation was estimated with a histological score based on villus epithelium adhesion, on villus sloughing and on crypt cell adhesion to the basal membrane. Antioxidant potential was estimated by measurement of total thiol functions (SH) and activities of glutathione-peroxidase (GSH-P), superoxide dismutase (SOD) and catalase. FC in flush improved preservation during the first 24h (p<0.01). Storage in FC appeared superior to UW for the first 24h (p<0.01). Oxygenation (100% O2) of the storage medium yielded superior results at 12h and 24h (p<0.01 and p<0.001 versus group [1] respectively). After 72h, SOD and catalase activities increased in groups [3] and [4], and SOD decreased in group [1] (p<0.05). SH progressively decreased in group [1] (p<0.05) and GSH-P increased at 24 and 48h in groups [3] and [4] (p<0.01). The increase of O2 in the perfusion flush or storage medium ameliorated the preservation status and protected the antioxidant potential of the small bowel.

Anti-ICAM-1 blockade reduces postsinusoidal WBC adherence following cold ischemia and reperfusion, but does not improve early graft function in rat liver transplantation

Background/Aim: The present in vivo study investigated the impact of a monoclonal antibody directed against the intercellular adhesion molecule-1 (ICAM-1) on initial microvascular reperfusion injury after liver transplantation. Methods: Orthotopic, syngeneic liver transplantation including arterial reconstruction was performed in male Lewis rats after 24 h graft storage in University of Wisconsin (UW) solution at 4°C. Animals received either an anti-ICAM-1 antibody ( n=7), an IgG 1 control antibody ( n=8) or saline only ( n=7). Hepatic microvascular alterations during the initial 90 min of reperfusion were assessed using intravital fluorescence microscopy. Early graft dysfunction was determined by analysis of bile flow. Results: After treatment with anti-ICAM-1 mAb, hepatic microvascular perfusion was found improved when compared with that of IgG 1- and saline-treated controls. In addition, anti-ICAM-1 mAb effectively reduced the number of permanently adherent white blood cells in postsinusoidal venules (284.4±59.1 mm −2 vs IgG 1: 371.9±26.7 mm −2 and saline: 431.8±46.4 mm −2; p<0.05). In contrast, the number of stagnant white blood cells in sinusoids was higher ( p<0.05) in liver grafts with blocked ICAM-1 (320.6±17.2 mm −2) compared with that of IgG 1- (215.2±11.1 mm −2) and saline-treated controls (226.4±14.0 mm −2). Measurement of hepatic uptake of fluorescent-labeled latex particles did not reveal significant differences in phagocytic activity. Finally, bile flow also did not differ between the three groups studied. Conclusion: Together these results indicate that ICAM-1 is involved in the process that mediates white blood cells adherence in postsinusoidal venules, whereas in hepatic sinusoids other mechanisms apart from ICAM-1-mediated white blood cells adherence seem to be fundamental for posttransplant white blood cells accumulation. Our data further suggest that white blood cells adherence in postsinusoidal venules via ICAM-1 does not make a major contribution to the pathogenesis of early cold ischemia/reperfusion injury after liver transplantation.

CLINICAL APPLICATION OF THE TWO-LAYER [UNIVERSITY OF WINSONSIN SOLUTOIN (UW)/PERFLUOROCHEMICAL (PFC)] METHOD OF PANCREAS PRESERVATION BEFORE TRANSPLANTATION.

435 Pancreas preservation after procurement is essential for pancreas transplantation. Currently, simple storage in University of Wisconsin (UW) solution is the standard preservation method, however, it can only preserve the human pancreas for less than 30 hours. If we can preserve the pancreas longer not only we will have more time to prepare for organ transplantation but also the donor organ pool will increase and alleviate shortages. In previous studies, the two-layer method for pancreas preservation has been demonstrated to be superior to UW storage using canine models. The purpose of this study is to confirm the clinical significance of the two-layer method compared with UW storage alone before pancreas transplantation. Methods. From October 1997 to November 1998 we performed 54 pancreas transplants using pancreases from local OPO's. Pancreases were placed in the two-layer method in 10 cases and UW alone in 44 cases prior to transplantation. We compared the condition of grafts at the time of reperfusion, graft function and complications 3 months post-transplantation. In addition, after preservation we collected preservation fluid to examine the bacterial contamination. Results. There was no significant difference between the demographics of donors and recipients as well as no bacterial contamination in any of the donor organs. Total cold ischemia time was 16.5 ± 5.0 hr in the two-layer group and 18.1 ± 4.7 hrs in the UW group with no statistical difference. At the time of reperfusion no grafts in the two-layer group were edematous compared with 10 of 43 (23.3%) in the UW group. All grafts in two-layer group remained soft, compared with 38 of 43 (88.3%) in the UW group at the time of reperfusion. Seven of 10 (70%) grafts obtained the best overall score in the two-layer group and 24 of 42 (57.1%) in the UW group. Nine of 10 (90%) patients in the two-layer group were insulin independent during hospitalization compared with 31 of 44 (70.5%) in the UW group. No patients in two-layer group rejected their grafts compared with 7 of 44 (15.9%) in the UW group. Conclusions. The two-layer method of pancreas preservation could be beneficial in clinical transplantation being that it was superior to UW in both morphological and functional assessment.

Expression of Stress Proteins, Adhesion Molecules, and Interleukin-8 in Endothelial Cells after Preservation and Reoxygenation

Endothelial activation is a central feature of preservation-induced allograft injury. The present study aims at a quantitative assessment of stress proteins, adhesion molecules, and interleukin-8 in a cell culture-based model of organ preservation. Human umbilical vein endothelial cells were exposed to cold, hypoxic storage in University of Wisconsin (UW), histidine-tryptophane-ketoglutarate (HTK), and EuroCollins solutions for 8 h with subsequent rewarming/reoxygenation (rew/reox) for 1 and 4 h. A cell-based ELISA was designed for detection of heat shock proteins (HSP) 60 and 70, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (ELAM-1). Immunohistochemical staining was performed for comparison. Interleukin-8 was quantified by ELISA. HSP 70 was expressed after cold storage in HTK and EuroCollins solution and after rew/reox in all groups. A constitutive expression of HSP 60 was observed with further upregulation after rew/reox following cold storage in all experimental groups. ICAM-1 was clearly upregulated, but VCAM-1 showed only weak expression after cold storage and rew/reox. ELAM-1 was detectable in minimal amounts after cold storage but was considerably upregulated after 4 h of rew/reox. A significant increase of interleukin-8 release could be found after 4 h of rew/reox following storage in EuroCollins solution. Expression of stress proteins can be considered as a new parameter of preservation-associated endothelial activation. Apart from possible protective effects, allograft vasculopathy could be in part a consequence of the antigeneic potential of heat shock proteins connected with effects caused by adhesion molecules and inflammatory cytokines.

Reduction of hepatic ischemia/reperfusion injury by a soluble P-selectin glycoprotein ligand-1.

The authors' goal was to determine the effects of specific binding and blockade of P- and E-selectins by a soluble P-selectin glycoprotein ligand-1 (PSGL-1) in rat models of hepatic in vivo warm ischemia and ex vivo cold ischemia. The authors also sought to determine the effect of selectin blockade on isograft survival in a syngeneic rat orthotopic liver transplant model. Ischemia/reperfusion (I/R) injury is a major factor in poor graft function after liver transplantation, which may profoundly influence early graft function and late changes. It is hypothesized that I/R injury leads to the upregulation of P-selectin, which is then rapidly translocated to endothelial cell surfaces within 5 minutes of reperfusion of the liver, initiating steps leading to tethering of polymorphonuclear neutrophil leukocytes to the vascular intima. Local production by leukocytes of interleukin-1, tumor necrosis factor-alpha, or both induces P-selectin expression on the endothelium and continues the cascade of events, which increases cell adherence and infiltration of the organ. To examine directly the effects of selectins in a warm hepatic I/R injury model, 100 microg of PSGL-1 or saline was given through the portal vein at the time of total hepatic inflow occlusion. The effects of PSGL-1 in cold ischemia were assessed using an isolated perfused rat liver after 6 hours of 4 degrees C storage in University of Wisconsin (UW) solution, with or without the instillation of PSGL-1 before the storage. To evaluate the effect of selectin blockade on liver transplant survival, syngeneic orthotopic liver transplants were performed between inbred male Sprague-Dawley rats after 24 hours of cold ischemic storage in UW solution. A separate group of animals received two doses of 100 microg of PSGL-1 through the portal vein before storage and before reperfusion of the transplanted liver. Recipient survival was assessed at 7 days, and the Kaplan-Meier product limit estimate method was used for univariate calculations of time-dependent recipient survival events. In an in vivo warm rat liver ischemia model, perfusion with PSGL-1 afforded considerable protection from I/R injury, as demonstrated by decreased transaminase release, reduced histologic hepatocyte damage, and suppressed neutrophil infiltration, versus controls (p < 0.05). When cold stored livers were reperfused, PSGL-1 reduced the degree of hepatocyte transaminase release, reduced neutrophil infiltration, and decreased histologic hepatocyte damage (p < 0.05 vs. UW-only controls). On reperfusion, livers treated with PSGL-1 demonstrated increased portal vein blood flow and bile production (p < 0.05 vs. UW-only controls). In addition, 90% of the rats receiving liver isografts stored in UW solution supplemented with PSGL-1 survived 7 days versus 50% of those whose transplanted syngeneic livers had been stored in UW alone (p < 0.05). Selectins play an important role in I/R injury of the liver. Early modulation of the interaction between P-selectin and its ligand decreases hepatocyte injury, neutrophil adhesion, and subsequent migration in both warm and cold rat liver ischemia models. In addition, the use of PSGL-1 before ischemic storage and before transplantation prevents hepatic injury, as documented by a significant increase in liver isograft survival. These findings have important clinical ramifications: early inhibition of alloantigen-independent mechanisms during the I/R damage may influence both short- and long-term survival of liver allografts.

Investigation of a primary requirement of organ preservation solutions: supplemental buffering agents improve hepatic energy production during cold storage.

This study was designed to investigate the effects of a modified University of Wisconsin (UW) solution supplemented with one of four buffering agents (histidine, bicine [N,N-bis(2-hydroxyethyl)glycine], tricine [N-tris(hydroxymethyl)methylglycine], and Tris) on liver metabolism during cold ischemic storage. Rat livers were flushed and stored for a maximum period of 24 hr at 4 degrees C, and tissue energetics, substrate, and anaerobic end-products were assessed; the group exhibiting the best results during storage was recovered in a 60-min period of warm reperfusion. Relative buffering capacities of the experimental solutions (measured over physiological pH range, in mM H+/L) were: UW, 4.1; histidine+UW, 9.8; Tris+UW, 19.0; bicine+UW, 22.5; tricine+UW, 26.8. In the UW group, ATP levels dropped rapidly over the first 4 hr; 1.0 micromol/g (40% of initial) remained after 4 hr of storage. By 2 hr, ATP levels in bicine- and tricine-treated groups were 0.5 and 1.1 micromol/g greater than in the UW-stored livers and by 10 hr, ATP in bicine-treated livers was twofold that of the control (UW) group. Total adenylate levels also reflected a superior elevation of cellular energetics; even after 24 hr, quantities were 1.4 and 2.0 micromol/g higher than the UW group in bicine- and histidine-supplemented organs. The increase in energetics occurred as a result of increased flux through the major anaerobic energy-producing pathway, glycolysis. The glycolytic rate was significantly greater at storage times > 10 hr with solutions supplemented with bicine, histidine, and tricine. Final values for net lactate accumulation over the entire 24-hr storage period were: UW, 10.1 micromol/g; histidine, 14.3 micromol/g; bicine, 15.2 micromol/g; tricine, 13.8 micromol/g. Activities of glycogen phosphorylase revealed that the activity of this enzyme dropped by 50% within 2 hr of storage in UW. However, histidine and bicine supplementation resulted in a substantial elevation of phosphorylase "a" over 4 hr and 10 hr, respectively. The best buffer of the four examined in this study was bicine; energetics, glycolytic flux, and patterns of adenylate regeneration upon reperfusion were markedly superior to modified UW solution. The results of this study suggest that supplementing the "gold standard" UW solution with an additional buffering agent (in order of efficacy: bicine>tricine>histidine) may improve the metabolic status of livers during clinical organ retrieval/storage.

Respiratory defect as an early event in preservation-reoxygenation injury of endothelial cells.

Characterization of preservation injury in endothelial cells has been primarily accomplished by measurement of cell viability. To analyze early events and cellular mechanisms of preservation-reoxygenation injury, we developed high-resolution respirometry for the study of mitochondrial function in endothelial cells, to provide a quantitative marker for sublethal stress. Cultured human umbilical vein endothelial cells were stored for 4 and 8 hr at 4 degrees C under an atmosphere of 95% N2 and 5% CO2 in University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions. Respiration of suspended cells, measured after reoxygenation in growth medium at 37 degrees C, was significantly reduced in all treatments in comparison to controls not subjected to cold preservation. In contrast, trypan blue staining was unchanged after 4 hr of preservation and was significant only after 8 hr. After 8 hr of cold storage in UW and HTK solutions, respiration was 64+/-5% and 49+/-6%, respectively, of controls (46.5+/-3.3 pmol O2 x s(-1 x 10(-6) cells), indicating significantly better protection by UW solution than HTK solution. A titration regimen with substrate (succinate), uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), and inhibitors of complexes I and III (rotenone and antimycin A) resulted in identical respiratory response patterns in all treatments. The plasma membrane remained impermeable to succinate. Inner mitochondrial membrane function was preserved as indicated by a constant relative increase of respiration after uncoupling. These results demonstrate that loss of catalytic capacity for respiration constitutes an early event in preservation-reoxygenation injury, whereas membrane damage is not a primary defect. Respirometric evaluation of sublethal cell injury and localization of cell damage may provide selective guidelines for further optimization of strategies in organ preservation.

Intravital studies on beneficial effects of warm Ringer's lactate rinse in liver transplantation

Abstract This quantitative in vivo fluorescence microscopy study investigated the impact of warm versus cold Ringer's lactate (RL) graft rinse on various microvascular manifestations of ischemia-reperfusion injury after liver transplantation in the rat. Syngeneic orthotopic liver transplantation, including arterial revascularization, was performed in male Lewis rats following 24 h of cold storage in University of Wisconsin (UW) solution. In one group (n = 8) liver grafts were rinsed with 4 °C (cold) RL, whereas in the other group (n= 8) grafts were rinsed with 37 °C (warm) RL immediately prior to revascularization. Hepatic microvascular perfusion, leukocyte-endothelium interaction, and Kupffer cell activation were quantified 30–90 min after graft reperfusion by direct visualization with intravital fluorescence microscopy. Moreover, biliary excretory graft function was analyzed by determination of bile flow and bile salt excretion during the first 90 min after reperfusion. Compared to grafts rinsed with cold RL, acinar and sinusoidal perfusion were found to be significantly increased after rinsing the grafts with warm RL. The amount of non perfused acini declined from 18.1 %± 4.0 % to 7.4 %± 1.6 % (P < 0.05), and the total percentage of perfused sinusoids increased from 80.1 ± 1.4 to 88.4 ± 1.2 (P < 0.001) after cold and warm rinse, respectively. After rinsing the graft with warm RL, WBC adherence in sinusoids and especially in postsinusoidal venules decreased significantly by 28 % (P < 0.001) and 33 % (P < 0.001), respectively. Kupffer cell activation was markedly reduced after rinsing with RL at 37 °C, as indicated by a decelerated adherence of latex particles injected 80 min after reperfusion. Excretory graft function was dramatically increased following warm RL rinse during the 90-min observation period. Bile flow was enhanced from 1.04 ± 0.5 to 3.9 ± 0.8 ml/100 g liver per 90 min (P < 0.01), with a parallel rise in bile salt excretion from 24.3 ± 5.8 to 128.0 ± 19.8 mmol/ 100 g liver per 90 min (P < 0.05) when compared to cold RL. These data strongly suggest that rinsing liver grafts with warm RL prior to reperfusion represents a simple and inexpensive way to reduce the incidence of primary graft failure secondary to ischemia and reperfusion injury in liver transplantation. The results of this study were partly presented at the International Symposium on Transplantation of Abdominal Organs in Essen, Germany, in November 1992 and published in Transplantation Proceedings in 1993.

Intravital studies on beneficial effects of warm Ringer's lactate rinse in liver transplantation.

This quantitative in vivo fluorescence microscopy study investigated the impact of warm versus cold Ringer's lactate (RL) graft rinse on various microvascular manifestations of ischemia-reperfusion injury after liver transplantation in the rat. Syngeneic orthotopic liver transplantation, including arterial revascularization, was performed in male Lewis rats following 24 h of cold storage in University of Wisconsin (UW) solution. In one group (n = 8) liver grafts were rinsed with 4 degrees C (cold) RL, whereas in the other group (n = 8) grafts were rinsed with 37 degrees C (warm) RL immediately prior to revascularization. Hepatic microvascular perfusion, leukocyteendothelium interaction, and Kupffer cell activation were quantified 30-90 min after graft reperfusion by direct visualization with intravital fluorescence microscopy. Moreover, biliary excretory graft function was analyzed by determination of bile flow and bile salt excretion during the first 90 min after reperfusion. Compared to grafts rinsed with cold RL, acinar and sinusoidal perfusion were found to be significantly increased after rinsing the grafts with warm RL. The amount of nonperfused acini declined from 18.1% +/- 4.0% to 7.4% +/- 1.6% (P < 0.05), and the total percentage of perfused sinusoids increased from 80.1 +/- 1.4 to 88.4 +/- 1.2 (P < 0.001) after cold and warm rinse, respectively. After rinsing the graft with warm RL, WBC adherence in sinusoids and especially in postsinusoidal venules decreased significantly by 28% (P < 0.001) and 33% (P < 0.001), respectively. Kupffer cell activation was markedly reduced after rinsing with RL at 37 degrees C, as indicated by a decelerated adherence of latex particles injected 80 min after reperfusion. Excretory graft function was dramatically increased following warm RL rinse during the 90-min observation period. Bile flow was enhanced from 1.04 +/- 0.5 to 3.9 +/- 0.8 ml/100 g liver per 90 min (P < 0.01), with a parallel rise in bile salt excretion from 24.3 +/- 5.8 to 128.0 +/- 19.8 mmol/ 100 g liver per 90 min (P < 0.05) when compared to cold RL. These data strongly suggest that rinsing liver grafts with warm RL prior to reperfusion represents a simple and inexpensive way to reduce the incidence of primary graft failure secondary to ischemia and reperfusion injury in liver transplantation.

Glyoxalase II and glutathione levels in rat liver mitochondria during cold storage in Euro-Collins and University of Wisconsin solutions.

Glutathione and glyoxalase II levels were evaluated in cytosolic and mitochondrial compartments of rat liver after 7 and 24 hr of cold storage in University of Wisconsin (UW) and Euro-Collins solutions. 1-4 A similar time-dependent depletion of cytosolic glutathione up to about 60% of control values was observed in both Euro-Collins and UW solutions. Cytosolic glyoxalase II showed activity oscillations in livers stored in Euro-Collins but not in UW. Mitochondrial glutathione and glyoxalase II were severely depleted soon after 7 hr of cold storage in Euro-Collins, whereas the same parameters did not change in liver stored in UW after 24 hr. UW is confirmed to be the most suitable solution for liver cold storage and we conclude that mitochondrial glutathione and glyoxalase II can be important parameters in assessing mitochondrial and cell function.

Administration of Atracurium during Reperfusion of Rat Livers after 21 H of Cold Ischaemic Storage in Different Solutions

RECKENDORFER, H.; SPERLICH, M.; BURGMANN, H.; WEINDLMAYR-GOTTEL, M.; SPIECKERMANN, P. G.; SCHWARZ, S. Author Information

Administration of atracurium during reperfusion of rat livers after 21 h of cold ischaemic storage in different solutions

The pharmacokinetics of atracurium are not altered by impaired hepatic function. The drug is therefore used widely in liver transplant patients. In previous work on the hepatotoxic effects of atracurium in an isolated, perfused rat liver model, we could not detect biochemical (release of lactate dehydrogenase or aspartate aminotransferase) or histological evidence of liver cell damage, except a reduction in hepatic tissue ATP content. In the present study, rat livers were reperfused with Krebs-Henseleit bicarbonate buffer with or without atracurium after 21 h of cold ischaemic storage in University of Wisconsin (UW), Bretschneider's HTK or Euro-Collins solution. UW-protected livers showed a complete restoration of ATP, total adenine nucleotides and energy charge during reperfusion, but the addition of atracurium diminished the regeneration capacity to about 50%. The energy charge (an index for determination of liver viability) was also reduced markedly.

EVALUATION OF SOLUTIONS FOR SMALL INTESTINAL PRESERVATION

A number of organ preservation solutions have been formulated to slow the inevitable progression of ischemic injury, thus prolonging the storage time between removal and implantation. As adenine nucleotide content has been shown to correlate with the functional recovery of transplanted livers and hearts, this study investigated the effects of 24 hr of storage in three preservation solutions, saline (SA), Euro-Collins (CO), and University of Wisconsin (UW) on adenine and nicotinamide adenine nucleotides and inosine content of the rat small intestine. Significant biochemical differences were found between segments as early as after the initial perfusion when the inosine content was higher in UW-perfused than CO- or SA-perfused segments. After 2 hr of storage in CO solution and after 6 and 24 hr in both CO and UW solutions, the ATP content was higher than in SA-stored segments. In addition to inosine, which was significantly higher at all time points for UW-stored segments, the AMP and total adenine nucleotide content of UW-stored segments at 24 hr was significantly higher than SA- or CO-stored segments. After 24 hr of storage, those segments stored in UW were able to utilize significantly more oxygen than SA-stored. These data provide biochemical evidence supporting the advantages of CO and UW storage solutions over SA for preservation of small intestine segments.

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.

Microcirculatory disturbances and leucocyte adherence in transplanted livers after cold storage in Euro-Collins, UW and HTK solutions

Integrity of the hepatic microcirculation and maintenance of endothelial cell viability are critical components in preventing primary non-function after liver transplantation. Therefore, hepatic microcirculation and leucocyte-endothelial interaction were studied in rat livers stored for 1 h in Euro-Collins (EC), University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) solutions and subsequently transplanted. One hour after transplantation surgery, the livers were exposed under an intravital fluorescence microscope. After injection of the leucocyte marker acridine orange (1 mumol/kg), six pericentral fields were observed for 30 s and experiments were recorded continuously. The percentage of perfused sinusoids was reduced in the livers in the EC group (82.9%) in contrast to the UW (93.2%) and HTK groups (91.0%). Livers in the EC group showed a reduction in the diameters of pericentral sinusoids (7.3 +/- 0.2 microns; mean +/- SEM) compared with the UW group (9.5 +/- 0.2 microns; P less than 0.05) and HTK group (10.2 +/- 0.8 microns; P less than 0.05), indicating substantial cell swelling in livers stored in EC solution. Permanent adherence of leucocytes was most frequently observed in the EC group (33.5 +/- 1%), while this phenomenon was less pronounced in the UW group (14.5 +/- 1.1%; P less than 0.05) and HTK group (16.3 +/- 0.7%; P less than 0.05). Conversely, temporary adherence of leucocytes was reduced in the EC group (19.7 + 1.3%) compared with the UW group (30.5 + 2.1%) and the HTK group (34.4 + 0.8%).(ABSTRACT TRUNCATED AT 250 WORDS)

Microcireulatory disturbances and leucocyte adherence in transplanted livers after cold storage in Euro-Collins, UW and HTK solutions

Abstract. Integrity of the hepatic microcirculation and maintenance of endothelial cell viability are critical components in preventing primary non-function after liver transplantation. Therefore, hepatic microcirculation and leucocyte-endothelial interaction were studied in rat livers stored for 1 h in Euro-Collins (EC), University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) solutions and subsequently transplanted. One hour after transplantation surgery, the livers were exposed under an intravital fluorescence microscope. After injection of the leucocyte marker acridine orange (1 μmol/kg), six pericentral fields were observed for 30 s and experiments were recorded continuously. The percentage of perfused sinusoids was reduced in the livers in the EC group (82.9%) in contrast to the UW (93.2%) and HTK groups (91.0%). Livers in the EC group showed a reduction in the diameters of pericentral sinusoids (7.3 ± 0.2μm; mean ± SEM) compared with the UW group (9.5 ± 0.2μm; P < 0.05) and HTK group (10.2 ± 0.8 μm; P < 0.05), indicating substantial cell swelling in livers stored in EC solution. Permanent adherence of leucocytes was most frequently observed in the EC group (33.5 ± 1%), while this phenomenon was less pronounced in the UW group (14.5 + 1.1%; P < 0.05) and HTK group (16.3 ± 0.7%; P <0.05). Conversely, temporary adherence of leucocytes was reduced in the EC group (19.7 + 1.3%) compared with the UW group (30.5 + 2.1%) and the HTK group (34.4 + 0.8%). Microcireulatory failure and cell swelling in the EC group might be due to the lack of osmotic substances or oxygen radical scavengers included in UW (allopurinol, glutathione) and HTK (mannitol) solutions. In conclusion, cold storage of livers in U W and HTK solutions results in better preservation of the microcirculation and prevention of adhesion of leucocytes after transplantation compared with the EC solution.

Evidence that cold preservation-induced microcirculatory injury in liver allografts is not mediated by oxygen-free radicals or cell swelling in the rat.

The critical injury of cold preservation is to the hepatic microcirculation. Oxygen free radical injury and cell swelling have been proposed to be causes of allograft failure, and new solutions such as Marshall's isotonic citrate and University of Wisconsin (UW) solutions were designed to prevent cell swelling and free radical injury. Experiments were done to determine whether Marshall's solution and UW solution protect the microcirculation, and whether they do so by preventing cell swelling or free radical-induced injury. To determine if the new solutions reduce sinusoidal lining cell injury, rat livers were examined after preservation at 4 degrees C in NaCl 0.9% and CaCl2 2 mM for 4 hr and 8 hr, in Collins' solution for 8 hr, and in both UW and isotonic citrate solutions for 8 hr and 16 hr. Next, the role of cell swelling in preservation injury was studied by storing livers in hypotonic solutions that accelerate liver weight gain, and in a choline chloride-based preservation solution. Finally, to evaluate the role of active oxygen species, SOD, catalase, and allopurinol were added to preservation solutions. The effect of allopurinol alone was also studied. In a related study, sucrose was substituted for the free radical scavenger, mannitol, in isotonic citrate solution. All livers were studied by light microscopy after perfusion-fixation. Storage in UW and isotonic citrate solutions resulted in clear improvement in the morphology of the sinusoidal lining. Increasing the rate of liver weight gain by the use of hypotonic solutions did not accelerate the endothelial injury. Choline chloride-based solution prevented weight gain during preservation, but unlike UW or isocitrate solutions it did not retard the microcirculatory injury. After preservation in the presence of SOD and catalase, or allopurinol, no improvement in the defined morphological features of the endothelial injury was noted when compared with respective controls; nor was the benefit of isotonic citrate solution lessened by the removal of the free radical scavenger mannitol. We conclude that microvascular injury produced by cold injury is due neither to free radical-mediated injury nor to cell swelling. As both UW and isotonic citrate solutions provide significant protection to the microcirculation, they must do so by a yet-undetermined mechanism.