Comparing the effects of Dextran 70 and Hydroxyethyl starch in an intestinal storage solution

IntroductionOur lab has developed a novel strategy for intestinal preservation involving the intraluminal delivery of a nutrient-rich preservation solution. The aim of this study was to compare the effectiveness of two impermeant agents for use in our solution: Dextran 70 (D70; Mw = 70 kDa) and Hydroxyethyl starch (HES; Mw = 2200 kDa).MethodsRat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a ‘backtable’ intraluminal flush with: UW solution (group 1, UW), or an amino acid-based nutrient-rich preservation solution (AA solution) containing either 5% D70 (group 2, AA-D70) or HES (group 3, AA-HES). Tissue samples (n = 6) were taken at 2, 4, 8, and 12 h cold storage; histology, energetic, end-product, and oxidative parameters were assessed. In separate groups (n = 4), D70 and HES were fluorescently labeled with fluorescein isothiocyanate (FITC) in order to directly observe mucosal penetration of the starch and dextran.ResultsOver the 12 h storage time-course, direct visualization of the fluorescently labeled D70 showed penetration of the mucosal layer as early as 2 h and progressively continued to do so throughout the 12 h period. In contrast, HES did not cross the mucosal barrier and remained captive within the lumen. As time of storage progressed, grade of injury increased in all groups, however, at 4 and 12 h the AA-HES treated tissues exhibited significantly less injury compared to UW and AA-D70, P < 0.05. AA-HES group showed on moderate villus clefting (median grade 2; P < 0.05) while the AA-D70 group exhibited complete villus denudation (grade 4) and the UW group had extensive injury into the regenerative cryptal regions (grade 6). Metabolic parameters revealed a preferential maintenance of ATP and Energy Charge; increases in lactate, alanine and ammonium supported the involvement of aerobic and anaerobic pathways for energy production.ConclusionThe results of this study challenge the idea that oncotic support is not a fundamental requirement of static organ storage. Furthermore, our data suggests that HES is an effective oncotic agent for use in our intraluminal nutrient-rich preservation solution, while Dextran 70 is not.     

Optimizing the concentration of hydroxyethylstarch in a novel intestinal-specific preservation solution

Introduction

Our lab has developed an effective nutrient-rich solution that facilitates energy production and control of oxidative stress during static cold storage of the intestine; however, the requirement for oncotic agents, such as hydroxyethylstarch (HES), has not been evaluated. This study investigated the effectiveness and requirement for HES in an intraluminal preservation solution during a clinically relevant period of cold storage.

Methods

Rat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a ‘back table’ intraluminal flush with a nutrient-rich preservation solution containing varying amounts of HES (n = 6 per group): Group 1, 0%; Group 2, 2.5%; Group 3, 5%; Group 4, 10%. Energetics, oxidative stress, and morphology were assessed over a 24 h time-course of cold storage.

Results

Overall, the 5% HES solution, Group 3, demonstrated superior energetic status (ATP and total adenylates) compared to all groups, P < 0.05. Malondialdehyde levels indicated a reduction in oxidative stress in Groups 3 and 4 (P < 0.05). After 12 h, median modified Parks’ grades for Groups 2 and 3 were significantly lower than Groups 1 and 4, P < 0.05.

Conclusion

Our data suggests that when employing an intraluminal preservation solution for static organ storage, oncotic support is a fundamental requirement; 5% HES is optimal.     

The influence of short-term fasting on the quality of small bowel graft preservation

INTRODUCTION: Donor nutritional status may be a determinant of small bowel (SB) quality following storage. In this study, we investigated the effect of donor nutritional status and a proven nutrient-rich preservation solution on graft quality following cold storage. METHODS: Rats were fasted (12-14 h) or non-fasted. SB (n=6) was flushed vascularly with modified University of Wisconsin (UW) solution and flushed luminally with UW or an amino acid-rich (AA) solution as follows: Fasted. UWV, none; UWL, UW solution; AAL, AA solution. Non-fasted. UWV, none; UWL, UW solution; AAL, AA solution. Energetics, peroxidation (malondialdehyde; MDA), glutathione and histology were assessed over 24 h at 4 degrees C. RESULTS: Energetics (ATP, ATP/ADP, and energy charge) were significantly higher in AAL (fasted and non-fasted) groups than other groups. However, there were no differences in energetics parameters between fasted and non-fasted animals in all groups. MDA was higher in fasted groups than non-fasted tissues; interestingly, AAL values were up to 10-fold lower than other groups. Higher glutathione levels were detected in non-fasted AAL tissues. Mucosal integrity was markedly superior in luminally treated tissues (UWL and AAL) in fasted and non-fasted states. Most noteably, AAL tissues from fasted animals exhibited grade 2 injury (villus clefting), whereas normal mucosa was observed in non-fasted tissues (grade 0). CONCLUSION: Luminal flushing and a nutrient-rich preservation solution improve energetics, oxidative stress, and mucosal integrity during storage. Poorer donor nutritional status does not affect energetics throughout storage, but causes mucosal injury as a result of increased oxidative stress, even after a brief period of donor fasting.     

Hyperbranched Polyglycerol as a Colloid in Cold Organ Preservation Solutions

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation.     

Improve islet yields and quality when clinical grade pancreata are preserved by the two-layer method

Background: Research grade pancreata preserved by the two-layer method (TLM) yield significantly greater numbers of islets than organs stored with University of Wisconsin solution (UW). The goal of this study was to determine whether this would hold true for pancreata that meet selection criteria for clinical grade organs. Methods: Pancreata were chosen based upon a pre-defined set of criteria used for selecting clinical grade pancreata. Thirteen of these organs were preserved in UW and five pancreata were preserved by the TLM. Islets were isolated and evaluated according to the Edmonton protocol. Results: The average preservation time was significantly longer for organ preserved with TLM (9.5 + 2.0 h) as compared to UW (5.8 + 0.6 h, p = 0.015). The pancreata of TLM group resulted in a significant increase in islet yields (3588 ± 500 vs. 2536 ± 312 IE/g pancreas, p<0.05). Visual scoring of islets indicated that islets were better from TLM group (8.3 ± 0.3 vs. 7.3 ± 0.2), and islet survival rates after culture were higher from organs stored with the TLM (87 ± 17 vs. 55 ± 7.4, p<0.05). Other parameters such as viability, insulin content, and stimulation index were similar between the two groups. All the preparations from the TLM group, but only 54% of preparations from the UW group, qualified for islet transplantation. The two recipients receiving islets from TLM group, daily insulin requirements were reduced and C-peptide levels were increased. Conclusion: Compared to storage with UW, exposure of pancreata to the TLM resulted in greater islet yields and improved quality of islets despite longer preservation period. Consequently, pancreata that meet clinical grade status should be preserved by the TLM prior to islet isolation.     

Creatine-loading preserves intestinal barrier function during organ preservation

We have developed a novel, intraluminal preservation solution that is tailored to the metabolic requirements of the intestine. This organ-specific solution addresses many of the problems associated with low temperature organ storage including energy, oxidative and osmotic stresses. However, conservation of energy levels remains one of the most difficult obstacles to overcome due to the inherent sensitivity of the mucosa to ischemia. Creatine-loading has become a popular and scientifically proven method of augmenting energy reserves in athletes performing anaerobic burst work activities. We hypothesized that if we could develop a method that was able to augment cellular energy levels, the structure and function of the mucosa would be more effectively preserved. The purpose of this study was to determine if creatine-loading is a feasible and effective strategy for preserving the intestine.Our data indicate that creatine loading has significant impact on energy levels during storage with corresponding improvements in mucosal structure and function. Both of our rodent models, a) continuous perfusion for 4 h and b) a single flush with our intraluminal preservation solution supplemented with 50 mM creatine, demonstrated significant improvements in creatine phosphate, ATP, Energy Charge and ATP/AMP following cold storage (P < 0.05). Notably, after 10 h creatine phosphate was 324% greater in Creatine-treated tissues compared to Controls (P < 0.05). Preferential utilization of glutathione in the Creatine group was effective at controlling oxidative injury after 10 h storage (P < 0.05). Improvements in barrier function and electrophysiology with creatine-treatment reflected superior mucosal integrity after 10 h storage; Permeability and Transepithelial resistance measurements remained at fresh tissue values. This was in stark contrast to Control tissues in which permeability rose to >300% of fresh tissue values (P < 0.005) and transepithelial resistance dropped by 95% (P < 0.005). After 10 h storage, Park's grading of histologic injury reflected extensive villus denudation (grade 4) in control tissues compared to healthy tissue (grade 0) in the Creatine group. This study demonstrates that a strategy of creatine supplementation of our intraluminal preservation solution facilitates the preservation of the intestinal mucosa during storage.     

Impact of normothermic preservation with extracellular type solution containing trehalose on rat kidney grafting from a cardiac death donor

Background

The aim of this study was to investigate factors that may improve the condition of a marginal kidney preserved with a normothermic solution following cardiac death (CD) in a model of rat kidney transplantation (RTx).

Methods

Post-euthanasia, Lewis (LEW) donor rats were left for 1 h in a 23°C room. These critical kidney grafts were preserved in University of Wisconsin (UW), lactate Ringer''s (LR), or extracellular-trehalose-Kyoto (ETK) solution, followed by intracellular-trehalose-Kyoto (ITK) solution at 4, 23, or 37°C for another 1 h, and finally transplanted into bilaterally nephrectomized LEW recipient rats (n = 4–6). Grafts of rats surviving to day 14 after RTx were evaluated by histopathological examination. The energy activity of these marginal rat kidneys was measured by high-performance liquid chromatography (HPLC; n = 4 per group) and fluorescence intensity assay (n = 6 per group) after preservation with UW or ETK solutions at each temperature. Finally, the transplanted kidney was assessed by an in vivo luciferase imaging system (n = 2).

Results

Using the 1-h normothermic preservation of post-CD kidneys, five out of six recipients in the ETK group survived until 14 days, in contrast to zero out of six in the UW group (p<0.01). Preservation with ITK rather than ETK at 23°C tended to have an inferior effect on recipient survival (p = 0.12). Energy activities of the fresh donor kidneys decreased in a temperature-dependent manner, while those of post-CD kidneys remained at the lower level. ETK was superior to UW in protecting against edema of the post-CD kidneys at the higher temperature. Luminescence intensity of successful grafts recovered within 1 h, while the intensity of grafts of deceased recipients did not change at 1 h post-reperfusion.

Conclusions

Normothermic storage with extracellular-type solution containing trehalose might prevent reperfusion injury due to temperature-dependent tissue edema.     

Beneficial effects of supplemental buffer and substrate on energy metabolism during small bowel storage

Successful preservation of small bowel (SB) is closely correlated with the maintenance of cellular energetics. This study was designed to assess the ability of a modified UW solution supplemented with buffer and glucose to facilitate ATP production during cold storage. In part A, rats SB (n = 4) were flushed vascularly as follows: Group 1, UW solution (control); Group 2, HUW solution (UW+90 mM histidine). Inclusion of histidine resulted in a >3-fold increase in buffering capacity over the pH range 7.4-6.8. Positive effects of histidine on ATP and energy charge were apparent after 4-10h storage. Examination of the key regulatory enzyme, Phosphofructokinase (PFK), reflected a sustained activation was over 1-4h in the HUW group only. In part B, groups were vascularly flushed as follows: Group 1, HUW solution (control); Group 2, Group 1+20mM glucose; and Group 3, Group 2+luminal flush. Elevated ATP and total adenylates over 2-10h in Group 3 compared to control were a direct consequence of improved glycolytic activity. This data supports the hypothesis that tissue energetics can be significantly improved during cold storage using a histidine-buffered UW solution supplemented with carbohydrate substrate.     

University of Wisconsin Solution with Trypsin Inhibitor Pefabloc Improves Survival of Viable Human and Primate Impure Islets During Storage

Background. Recent studies suggest that impure islets (islets which have not been isolated from exocrine tissue and other parts of the pancreas) have great potential for successful transplantation. The evidence that supports this view includes findings that embedded islets (islets surrounded by exocrine tissue) undergo less apoptosis, peripancreatic lymph nodes prevent recurrence of IDDM (insulin dependent diabetes mellitus), and that islet yields and insulin content decrease during the purification process. Improved protocols have also been developed to prevent allorejection of impure islets. Despite these promising results, the storage of impure islets remains difficult, and was a method sought to decrease storage losses. Methods. Storage methods of impure human and non-human primate islets were compared, using either culture media or University of Wisconsin solution (UW). The effects of trypsin inhibition using Pefabloc (Roche Molecular Biochemicals, Indianapolis, IN) during storage period were also examined. Results. Low temperature and inhibition of trypsin activity during storage of impure islets improved both islet yield and viability. It was found that using UW solution and trypsin inhibition allowed perfect preservation of viable impure islets up to 48h. A functional assay by glucose stimulation test showed these impure islet responded to glucose stimulation after 24h. Conclusion. The benefits of storing impure islets using UW solution and Pefabloc at low temperature have been established. This improved method of preserving impure islets makes this model of transplantation even more viable.     

The Efficacy of Antioxidants Administered during Low Temperature Storage of Warm Ischemic Kidney Tissue Slices

Accumulation of products of lipid peroxidation (malondialdehyde, conjugated dienes, lipid peroxides, and Schiff bases) was evaluated in rabbit kidney cortex slices made ischemic for 60 min followed by 18 h storage at 5°C in UW Na gluconate solution and 210 min normothermic reoxygenated incubation. In addition, the effect of adding Trolox (1 mM), deferoxamine (1 mM), and ascorbate (1 mM) as supplemental antioxidants to the UW gluconate solution was evaluated. Lipid peroxidation was slightly increased after hypothermic storage compared to slices subjected to ischemia alone but was not significantly different than ischemic slices during subsequent incubation at normothermia. The addition of either deferoxamine or Trolox to the storage solution substantially reduced lipid peroxidation both during hypothermic storage and subsequent to normothermic incubation. Ascorbate had a mild prooxidant effect as a sole additive to the UW gluconate solution but was clearly prooxidant when combined with either deferoxamine or Trolox. These results suggest that supplemental antioxidants added to the UW gluconate solution under conditions analogous to machine perfusion preservation have a potential role in reducing oxidative stress in kidney tissues harvested after warm ischemia and that hypothermia may be a valuable adjunct to resuscitative therapeutic regimens developed for salvage of ischemic kidneys for transplantation.     

A comparison of a sucrose-based solution with other preservation media for cold storage of isolated hepatocytes

A sucrose-based solution has been compared with other preservation solutions (University of Wisconsin (UW) solution and Marshall's citrate solution, with Dulbecco's medium as control) during hypothermic preservation of isolated rat hepatocytes for up to 72 h. Studies on the stability of liver cells at low temperature by exclusion of trypan blue dye and morphological appearance were conducted. During storage beyond 24 h, there was a clear difference between cells stored in Dulbecco's medium and Marshall's citrate and those stored in sucrose-based solution and UW solution, with the former storage groups showing many cells developing large membrane "blebs" and the latter storage groups maintaining a more typical morphology and developing only small membrane protrusions. Dye exclusion was higher in sucrose-based solution (48 h, 75 +/- 7%; 72 h, 65 +/- 6%) and UW solution (48 h, 72 +/- 5%; 72 h, 63 +/- 4%) than in Marshall's citrate (48 h, 31 +/- 5%; 72 h, 10 +/- 1%) and Dulbecco's medium (48 h, 8 +/- 2%; 72 h, 5 +/- 1%). These data suggest that sucrose-based solution should be investigated further as a less complex alternative solution for storage of isolated hepatocytes.     

Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions

Introduction

Endothelial barrier function is pivotal for the outcome of organ transplantation. Since hypothermic preservation (gold standard) is associated with cold-induced endothelial damage, endothelial barrier function may benefit from organ preservation at warmer temperatures. We therefore assessed endothelial barrier integrity and viability as function of preservation temperature and perfusion solution, and hypothesized that endothelial cell preservation at subnormothermic conditions using metabolism-supporting solutions constitute optimal preservation conditions.Methods: Human umbilical vein endothelial cells (HUVEC) were preserved at 4–37 °C for up to 20 h using Ringer's lactate, histidine–tryptophan–ketoglutarate solution, University of Wisconsin (UW) solution, Polysol, or endothelial cell growth medium (ECGM). Following preservation, the monolayer integrity, metabolic capacity, and ATP content were determined as positive parameters of endothelial cell viability. As negative parameters, apoptosis, necrosis, and cell activation were assayed. A viability index was devised on the basis of these parameters.Results: HUVEC viability and barrier integrity was compromised at 4 °C regardless of the preservation solution. At temperatures above 20 °C, the cells' metabolic demands outweighed the preservation solutions' supporting capacity. Only UW maintained HUVEC viability up to 20 °C. Despite high intracellular ATP content, none of the solutions were capable of sufficiently preserving HUVEC above 20 °C except for ECGM.Conclusion: Optimal HUVEC preservation is achieved with UW up to 20 °C. Only ECGM maintains HUVEC viability at temperatures above 20 °C.     

Microvascular changes of the liver preserved in UW solution. Pathological and immunohistochemical examination.

Rat livers preserved in University of Wisconsin (UW) solution for 24 h were compared with those preserved in Euro-Collins (EC) solution before and after liver transplantation using an immunohistochemical method. Tissue ATP and total tissue adenine nucleotide (TAN) were measured using HPLC. The levels of TAN in the UW group or the EC group were significantly low compared with the control group (no preservation) after 24-h storage. In the EC group, the levels of tissue adenine nucleotides (TAN) decreased 1 h after reperfusion and never reached control levels. In the UW group, the levels of TAN increased a little 1 h after reperfusion and increased more 3 h after reperfusion. After 24-h preservation, the expression of factor VIII-related antigen (FRA) in endothelial cells of central veins was weak in the EC group; in the UW group, FRA was clearly detected in these cells. After reperfusion, although severe endothelial cell damage to the central veins and numerous FRA-positive substances were observed in EC group, endothelial cells of central veins retained their normal structure and FRA-positive substances were rarely noted in the UW group. In both groups, no endothelial changes were detected in portal veins. From these results, it is concluded that UW solution prevents endothelial cell damage and microcirculatory injury in zone III during the preservation period resulting in prevention of initial graft nonfunction. Also, measurement of the TAN level after reperfusion is useful to predict the function of the graft.     

Hypothermic storage of coronary endothelial cells reduces nitric oxide synthase activity and expression

Preservation with University of Wisconsin (UW) solution has been implicated in coronary artery endothelial damage and loss of endothelium-dependent vasodilatation. Therefore, the objective of this study was to investigate the effect of this solution on basal nitric oxide (NO) release from porcine coronary endothelial cells (CEC). Cultures were exposed to cold (4 degrees C) storage in UW solution for 6, 8 and 12 h. Parallel cultures were incubated with control medium at 37 degrees C. After treatment, NO release was evaluated by nitrite production, a stable metabolite of NO. Activity of the constitutive endothelial nitric oxide synthase (eNOS) was measured by the conversion [3H]-l-arginine to [3H]-l-citrulline and eNOS protein expression by Western blotting. Nitrite production by control cells was augmented with increasing times of incubation, whereas no change was observed in those cultures preserved with UW solution. Activity of eNOS was significantly decreased compared to the respective control group by cold storage of cells for longer periods than 6 h. Such decrease was correlated with a diminished eNOS protein expression in CEC preserved with UW solution after 8- and 12-h storage. These results suggest that prolonged hypothermic storage of CEC with UW solution does not preserve basal NO release because of a certain loss of eNOS protein, which may contribute to the reported injury of heart transplants after long-term preservation.     

Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4^oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4^oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 ^oC). We find that there exists an optimum temperature (-4^oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.     

The functional effects of suppression of hypothermia-induced cell swelling in liver preservation by cold storage

It is known that cellular edema and functional impairment develop during anaerobic cold storage of organs. The extent of both is related to the storage time and the composition of the preservation solution used. We studied hypothermia-induced cell swelling and its effect on liver function after cold storage preservation with either Eurocollins (EC), a number of modified EC solutions in which glucose was replaced by various concentrations of raffinose, or UW solution. After 24 h storage, tissue swelling as determined by total tissue water (TTW) in rat liver tissue slices was most pronounced in slices incubated in Eurocollins, whereas the TTW was only moderately increased in slices stored in modified Eurocollins containing 90 to 120 mM raffinose. In contrast, slices incubated in UW solution had a TTW equal to normal rat liver tissue. Furthermore, intact rabbit livers preserved with Eurocollins had an increase in the whole organ weight, while there was no weight change after preservation with the modified solution containing 120 mM raffinose (M120). In contrast, a pronounced weight loss was observed after preservation with UW solution. After cold storage, the livers were reperfused for 2 h at 38 degrees C in an isolated perfusion circuit (IPL) with an acellular perfusate. Bile flow was significantly greater in livers preserved in M120 than in those preserved with the conventional Eurocollins. However, the bile flow in the livers stored in M120 was inferior to that in the livers preserved with UW solution, which in turn was equal to that in control livers. The release of alanine-aspartate-aminotransferase into the perfusate was higher in livers preserved with Eurocollins, with or without modification, than in the livers preserved with UW solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine improves hypothermic machine preservation of the liver

Background

Hypothermic machine preservation (HMP) is currently reconsidered as alternative to standard cold storage of organs from non-heart-beating donors. The present study was aimed at investigating the possible synergistic effect of HMP and the addition of dopamine to the circulating perfusate during preservation.

Methods

Cardiac arrest was induced in male Wistar rats (250–300 g) by phrenotomy. Thirty minutes later livers were flushed via the portal vein and subjected to 20 h of HMP at 5 ml/min at 4 °C. During HMP the preservation solution was equilibrated with 100% oxygen and dopamine was added at 0, 10, 50 or 100 μM (D0, D10, D50, D100; n = 6 resp.). Graft viability was assessed thereafter upon warm reperfusion in vitro for 2 h.

Results

During HMP, D50 and D100 significantly reduced hepatic release of ALT to about 50%. No influence of dopamine was found on vascular resistance, oxygen uptake or lactate production at any concentration. D50 significantly reduced enzyme release during reperfusion (∼50%), enhanced bile flow and oxygen consumption. D10 was less effective while D100 even rose enzyme release compared with D0.Enhanced oxygen free radical mediated lipid peroxidation (LPO), found in the tissue of D0 livers was significantly reduced by D50; D50 significantly abrogated molecular upregulation of vWillebrand factor upon reperfusion suggesting vascular protection of the endothelial cell.

Conclusion

Efficiency of HMP might be increased by stimulating livers with dopamine during ex vivo preservation, limiting vascular side effects and improving functional recovery upon early reperfusion.     

Contractile and morphological properties of hamster retractor muscle following 16 h of cold preservation

Introduction

Cold hypoxia is a common factor in cold tissue preservation and mammalian hibernation. The purpose of this study was to determine the effects of cold preservation on the function of the retractor (RET) muscle of the hamster in the non-hibernating state and compare these with previously published data (van der Heijden et al., 2000) [52] on the rat cutaneus trunci (CT) muscle.

Materials and methods

After cold storage (16 h at 4 °C), muscles were stimulated electrically to measure maximum tetanus tension (P₀) and histologically analyzed. The protective effects of addition of the antioxidants trolox and deferiprone and the calcium release inhibitor BDM to the storage fluid were determined.

Results

After storage, the twitch threshold current was increased (from 60 to 500 μA) and P₀ was decreased to 27% of control. RET morphology remained unaffected. RET muscle function was protected by trolox and deferiprone (P₀, resp., 43% and 59% of control). Addition of BDM had no effect on the RET.

Conclusions

The observed effects of cold preservation and of trolox and deferiprone on the RET were comparable to those on CT muscle function, as reported in a previously published study (van der Heijden et al., 2000) [52]. Both hamster RET and rat CT muscles show considerable functional damage due to actions of reactive oxygen species. In contrast to the CT, in the RET cold preservation-induced functional injury could not be prevented by BDM and was not accompanied by morphological damage such as necrosis and edema. This suggests that the RET myocytes possess a specific adaptation to withstand the Ca²⁺ overload induced by cold ischemia.     

Protective effects of a carbon monoxide-releasing molecule (CORM-3) during hepatic cold preservation

There is increasing evidence that carbon monoxide (CO), a signaling molecule generated during the degradation of heme by heme oxygenase-1 (HO-1) in biological systems, has a variety of cytoprotective actions, including anti-hypoxic effects at low temperatures. However, during liver cold preservation, a direct effect needs to be established. Here, we designed a study to analyze the role of CO, delivered via a carbon monoxide-releasing molecule (CO-RM) in the maintenance of liver function, and integrity in rats during cold ischemia/reperfusion (CI/R) injury. We used an isolated normothermic perfused liver system (INPL) following a clinically relevant model of ex vivo 48 h cold ischemia stored in a modified University of Wisconsin (UW) solution, to determine the specific effects of CO in a rat model. CO was generated from 50 μM tricarbonylchloro ruthenium-glycinato (CORM-3), a water-soluble transition metal carbonyl that exerts pharmacological activities via the liberation of controlled amounts of CO in biological systems. The physiological effects of CORM-3 were confirmed by the parallel use of a specific inactive compound (iCORM-3), which does not liberate CO in the cellular environment.CORM-3 addition was found to prevent the injury caused by cold storage by improving significantly the perfusion flow during reperfusion (by almost 90%), and by decreasing the intrahepatic resistance (by 88%) when compared with livers cold preserved in UW alone. Also, CORM-3 supplementation preserved good metabolic capacity as indicated by hepatic oxygen consumption, glycogen content, and release of lactate dehydrogenase. Liver histology was also partially preserved by CORM-3 treatment.

Conclusions

These findings suggest that CO-RM could be utilized as adjuvant therapeutics in UW solutions to limit the injury sustained by donor livers during cold storage prior to transplantation, as has been similarly proposed for the heart, and kidney.