Simple Cold Storage Research Articles

Low-temperature fluorometric technique for evaluating the viability of rat liver grafts after simple cold storage

Abstract. Time-dependent changes in the viability of rat liver graft during cold preservation with Euro-Collins solution were evaluated with NADH fluorometry. Correlation between the fluorometric analysis, 1-week survival rate after liver transplantation, and mitochondrial ATP synthesis activity in the early phase after transplantation was studied. Fluorometric study: Rat livers were preserved at 0d̀-4d̀C for 0–48 h in Euro-Collins solution and then reperfused for 15 min with oxygenated Krebs-Henseleit solution at 4d̀C. The amplitude (RxA) between the oxidized and the reduced steady-state NADH fluorometric trace and the velocity (RxV) of the trace were determined to evaluate the mitochondrial respiratory chain. The RxA and RxV remained at levels higher than 90% of control after 6-h preservation, while the R x A of the 9-h preservation group and the RxV of the 12-h preservation group decreased significantly compared with those of the control and the 6-h preservation group. Survival study: a 100% survival rate after transplantation was achieved in the 6-h preservation group, whereas the rates were 18.8% and 0% in the 9-and 12-h preservation groups respectively. These survival rates correlated closely with the time-dependent decrease of the fluorometric parameters. Study of mitochondrial phosphorylative activity and energy charge 3 h after transplantation: With fresh grafts, the decrease in hepatic energy charge after transplantation was reduced to 0.79 from the control value of 0.86 by a 30% increase in mitochondrial ATP synthesis ability. When the graft was preserved for 12 h, the energy charge dropped to 0.63 due to lack of the enhancement of ATP synthesis ability. The results of this study indicate a possibility of using fluorometric evaluation of the graft to predict post-transplantation mitochondrial ATP synthesis ability and survival rate.

The mechanism of action of the two-layer (Euro-Collins' solution/perfluorochemical) cold storage method in canine pancreas preservation.

To clarify the mechanism of action of a two-layer [Euro-Collins' solution (EC)/perfluorochemical (PFC)] cold storage method in the preservation of the pancreas, pancreatic viability and tissue concentrations of adenosine triphosphate (ATP) were examined in the canine model of pancreatic autotransplantation after preservation for 24 and 48 h by simple cold storage in EC (group 1), the two-layer, EC/PFC, method (group 2) and the two-layer, EC+2, 4 dinitrophenol (DNP)/PFC, method (group 3). DNP is an uncoupler of oxidative phosphorylation. Maintenance of normoglycemia for at least 5 days after transplantation was considered a successful preservation. After preservation for 24 h, the functional success rates of groups 1, 2 and 3 were 100% (4/4), 100% (5/5) and 80% (4/5) respectively. One of five dogs in group 3 died of a cause unrelated to the pancreas. ATP tissue concentrations in group 2 were significantly higher than in group 1 (7.47 +/- 0.47 micromol/g dry weight vs 1.41 +/- 0.53 micromol/g dry weight, P < 0.01) and ATP tissue concentrations in group 3 were significantly lower than in group 2 (1.25 +/- 0.37 micromol/g dry weight vs 7.47 +/- 0.47 micromol/g dry weight, P < 0.01). It was apparent that ATP was not an essential factor for successful 24-hour preservation of the canine pancreas in EC because all the pancreatic grafts except one of five grafts in group 3 remained viable after preservation for 24 h, regardless of ATP tissue concentrations. On the other hand, after preservation for 48 h, the functional success rates for groups 1, 2 and 3 were 0% (0/4), 100% (4/4) and 0% (0/3) respectively. ATP tissue concentrations in group 2 were significantly higher than in group 1 (7.91 +/- 1.21 micromol/g dry weight vs 1.21 +/- 0.31 micromol/g dry weight, P < 0.01) and ATP tissue concentrations in group 3 were significantly lower than in group 2 (0.61 +/- 0.07 micromol/g dry weight vs 7.91 +/- 1.21 micromol/g dry weight, P < 0.01). It was clear that preservation of the pancreas for 48 h was unsuccessful by simple cold storage in EC (group 1) and the two-layer method (group 2) made preservation for 48 h possible by increasing ATP tissue concentrations. However, DNP (group 3) inhibited the synthesis of ATP and the effectiveness of the two-layer method for 48-hour preservation of the pancreas. It was clear that maintenance of high ATP tissue concentrations during preservation was essential for the successful preservation of the canine pancreas in EC by the two-layer method for more than 48 h. We concluded that an adequate supply of oxygen to the pancreas during preservation by the two-layer method led to sufficient production of ATP to maintain cellular integrity and permitted the improvement of pancreatic preservation.

Successful 96-hour preservation of the canine pancreas.

We tested the preservation of the pancreas for 96 h by a modified two-layer (UW solution/perfluorochemical) cold storage method (group 1) in the canine model of pancreas autotransplantation and compared this with an original two-layer (Euro-Collins' solution/perfluorochemical) cold storage method (group 2) and simple cold storage method with UW solution (group 3). A graft was considered functioning if the dog had a normal blood glucose for at least 5 days after transplantation. The functional success rates after preservation for 72 h were 100%, 100% and 80% for groups 1, 2 and 3 respectively. On the other hand, the functional success rates for groups 1, 2 and 3 after preservation for 96 h were 75%, 0% and 0% respectively. The mean K value of 96-hour preserved grafts for group 1 at 2 weeks after transplantation was 1.52 +/- 0.30 compared with 1.98 +/- 0.48 before preservation. Biopsies of grafts from group 1 showed almost normal pancreatic architecture even after preservation for 96 h. In addition, biopsies of grafts preserved for 96 h in group 1 at 4 weeks after transplantation showed almost normal endocrine tissue with mild fibrosis of the exocrine tissue. This study demonstrated the possibility of preserving the pancreas for 96 h prior to transplantation.

Seventy-two-hour preservation of porcine liver by continuous hypothermic perfusion with UW solution in comparison with simple cold storage

Porcine livers preserved for 72 hr using continuous hypothermic perfusion (CHP) were studied in order to compare the effects of CHP on energy metabolism with those of simple cold storage (SCS). The livers of the CHP group were perfused in situ for 72 hr at 7°C with University of Wisconsin (UW) solution and FC-43 as an oxygen carrier, while those of the S1 group were stored ex situ for 48 hr at 4°C in UW solution, and those of the S2 group for 72 hr. They were then recirculated with human blood at 37°C for 2 hr for the evaluation of their viability. At the end of preservation, significantly higher levels of total adenine nucleotides (TAN) and energy charge (EC) were observed in the CHP group compared with the S1 and S2 groups. At 2 hr recirculation, the level of TAN was significantly higher in the CHP group than in the S1 and S2 groups. The EC level was also higher in the CHP group than in the S2 group. During recirculation, the ketone body ratio (acetoacetate/β-hydroxybutyrate) was higher in the CHP group than in the S2 group. The values in the CHP group were above 1.0 after 45 min recirculation. There were no significant differences in the pyruvate/lactate ratio and lactate level between the CHP and S1 groups. However, these values were significantly different from those in the S2 group. The present findings demonstrate that CHP using UW solution and the oxygen carrier was better able to preserve the energy metabolism of the porcine liver for 72 hr than 48-hr SCS in UW solution.

Effects of method of preservation on functions of livers from fed and fasted rabbits

Livers from fed, fasted (48 h) and glucose-fed rabbits were preserved for 24 and 48 h by either simple cold storage (CS) or continuous machine perfusion (MP) with the University of Wisconsin preservation solutions. After preservation liver functions were measured by isolated perfusion of the liver (at 37 °C) for 2 h. Fasting caused an 85% reduction in the concentration of glycogen in the liver but no change in ATP or glutathione. Glucose feeding suppressed the loss of glycogen (39% loss). After 24 h preservation by CS livers from fed or fasted animals were similar including bile production (6.2 ± 0.5 and 5.6 ± 0.4 ml/2 h, 100 g, respectively), hepatocellular injury (LDH release = 965 ± 100 and 1049 ± 284 U/liter), and concentrations of ATP (1.17 ± 0.15 and 1.18 ± 0.04 μmol/g, glutathione (1.94 ± 0.51 and 2.35 ± 0.26 μmol/g, respectively), and K:Na ratio (6.7 ± 1.0 and 7.7 ± 0.5, respectively). After 48 h CS livers from fed animals were superior to livers from fasted animals including significantly more bile production (5.0 ± 0.9 vs 2.0 ± 0.3 ml/2 h, 100 g), less LDH release (1123 ± 98 vs 3701 ± 562 U/liter), higher concentration of ATP (0.50 ± 0.16 vs 0.33 ± 0.07 (μmol/g) and glutathione (0.93 ± 0.14 vs 0.30 ± 0.13μmol/g), and a larger K:Na ratio (7.4 vs 1.5). Livers from fed animals were also better preserved than livers from fasted animals when the method was machine perfusion. The decrease in liver functions in livers from fasted animals preserved for 48 h by CS or MP was prevented by feeding glucose. Glucose feeding increased bile formation after 48 h CS preservation from 2.0 ± 0.3 (fasted) to 6.9 ± 1.2 ml/2 h, 100 g; LDH release was reduced from 3701 ± 562 (fasted) to 1450 ± 154 U/liter; ATP was increased from 0.33 ± 0.07 (fasted) to 1.63 ± 0.18 μmol/g; glutathione was increased from 0.30 ± 0.01 (fasted) to 2.17 ± 0.30 (μmol/g; and K:Na ratio was increased from 1.5 ± 0.9 to 5.3 ± 1.0. This study shows that the nutritional status of the donor can affect the quality of liver preservation. The improvement in preservation by feeding rabbits only glucose suggests that glycogen is an important metabolite for successful liver preservation. Glycogen may be a source for ATP synthesis during the early period of reperfusion of preserved livers.

THE ENHANCEMENT OF MYOCARDIAL PROTECTION THROUGH AN ACALCEMIC STORAGE SOLUTION CONTAINING NICARDIPINE, A POTENT CALCIUM CHANNEL BLOCKER

Adult rat ventricular myocytes were used as a model system for investigating the direct effects of nicardipine, a potent calcium-channel blocker, on preservation of the heart during periods of simple cold storage. Cells were stored at 4 degrees C for 24 hr with an acalcemic storage solution. The superfusate then was exchanged with hypoxic Tyrode solution (1.8 mmol/L CaCl2). After 2 hr of hypoxia at 20 degrees C, cells were reoxygenated and simultaneously warmed to 37 degrees C. The addition of nicardipine to both the storage solution and Tyrode solution resulted in the highest percentage of viable cells (70.5%). Using either the storage solution or Tyrode solution containing the compound, the percentages of viable myocytes were 51.9% and 52.2%, respectively. In the absence of the drug in either solution, the percentage was 38.0%. The effect of diltiazem, another potent calcium-channel blocker, was examined using the same experimental procedure. The addition of diltiazem to Tyrode solution elevated the viability of ventricular myocytes, but addition to the storage solution showed no cardioprotective effect. Moreover, the addition of 8-bromo cyclic GMP to the storage solution resulted in a cardioprotective effect. These results suggested that nicardipine exerts a direct effect on myocardial protection in hypothermic and acalcemic storage solution and that the pharmacological effect of nicardipine depends on a mechanism other than its calcium-channel blocking action.

Improved renal transplant preservation using a modified intracellular flush solution (PB-2)

A number of new intracellular renal flush solutions have been found to be more efficacious than Collins-2 (C-2) solution in extending organ viability during simple cold storage. However, the mechanism of action of these solutions remains poorly understood. To delineate better underlying intracellular mechanisms, we studied a modified, simple, hypothermic, intracellular (340 mOsm/kg) flush solution (PB-2). The development of PB-2 solution is based on the ability of some of its individual components to minimize ischemic adenine nucleotide (AN) catabolism and endothelial post "reperfusion injury." Preliminary results in 10 canine autorenal transplants show a significant (P less than 0.02) improvement in renal recovery and viability (recipient posttransplant inulin clearance and survival) after 50 h of cold storage compared with 10 canine kidneys similarly preserved using conventional C-2 flush solution. High performance liquid chromotography (HPLC) studies show a significant (P less than 0.01) loss of AN using C-2, while PB-2 was associated with regeneration of AN within 45 min of reperfusion. Magnetic resonance spectroscopy using phosphorus 31 (31P-MRS) showed more high energy phosphorus metabolites (phosphomonoester and nicotinamide-adenine-dinucleotide phosphate: P less than 0.001) at 50 h cold storage using PB-2 compared with C-2. Electron micrographs (EM) revealed normal microcapillary morphology for the PB-2 group; however, moderate vascular red and white blood cell clumping was observed in the C-2 group. Characterization of the basic preservation mechanisms by HPLC, 31P-MRS, and EM studies indicates that PB-2 solution enhances renal preservation by diminution of both reperfusion injury and the loss of intracellular high energy metabolites that are necessary for viability.

Effect of cold storage on tissue and cellular glutathione

One of the mechanisms thought to cause injury in preserved organs is the formation of oxygen free radicals. The cell is protected from oxidative stress by many defense mechanisms. A major defense mechanism involves glutathione and glutathione-dependent enzymes. During organ preservation by simple cold storage the loss of glutathione may sensitize the organ to free radical damage after transplantation. In this study we show that glutathione is depleted from the rabbit liver, kidney, and heart cold-stored (5 °C) for up to 72 h in the UW solution without glutathione. In the first 24 h kidney glutathione decreased to 84 ± 3% of control values, liver glutathione decreased to 49 ± 3% of control values, and heart glutathione decreased to 73 ± 3% of control values. After 48 h of storage the kidney and liver lost an additional 30 and 20%, respectively, whereas heart glutathione changed very little. By 72 h all three organs had lost more than 50% of the glutathione found in freshly obtained tissue. To determine if glutathione added to the UW solution can effectively prevent this loss of glutathione during preservation, hepatocytes were cold-stored for up to 72 h in a preservation solution with and without glutathione. We found that adding glutathione to the preservation solution slowed the rate of loss of glutathione from the cells. These data suggest that at hypothermia the cell may be permeable to GSH. Methods to suppress the loss of glutathione during preservation of organs may be an important factor in suppressing oxygen free radical injury.

Review article: improved preservation of the liver for transplantation

This paper reviews the development of the techniques used for liver preservation and describes their clinical use. Recent advances with the introduction of lactobionate based solutions for simple cold storage are described and illustrated by their effect on the Cambridge/King's College Hospital transplant programmes. Better preservation of the liver has simplified the logistics of the transplant procedure, improving organ usage and allowing increased sharing of livers for urgent or paediatric cases.

EXAMINATION OF THE ROLE OF THE IMPERMEANTS LACTOBIONATE AND RAFFINOSE IN A MODIFIED UW SOLUTION

Rat liver transplants were performed in order to assess the importance of the impermeant anion lactobionate and the trisaccharide raffinose on the effectiveness of a simplified variant of UW solution for liver preservation by simple cold storage. Rat livers were stored at 4 degrees C for 18, 24, 30, or 40 hr in a modified UW solution or in one of three variants of UW in which one of these impermeants was replaced by another more permeable agent. Using modified UW solution (solution A), 50% (5/10) of rats receiving livers that had been preserved for 30 hr survived for more than 1 week; with solution B, which differs from A in the replacement of raffinose by glucose, the 1-week survival was 60% (6/10) after 30-hr preservation. Solution C, which is identical to A except for the replacement of lactobionate by gluconate, gave 20% (2/10) survival rate after 30-hr preservation. However, using solution D, which is identical to A except for substitution of chloride for lactobionate, none (0/8) of the rats receiving liver preserved for 30 hr survived. These results suggest that the inclusion of lactobionate as a major anion plays a crucial role in the effectiveness of UW solution, whereas raffinose can be replaced by more permeant glucose without deleterious effect.

SEVENTY-TWO-HOUR PRESERVATION OF THE CANINE LIVER BY MACHINE PERFUSION

The UW solution effectively preserves the dog liver for up to 48 hr by simple cold storage. This solution contains lactobionate as the primary impermeant. Another solution developed for machine perfusion of the kidney is similar to the UW solution but contains gluconate in place of lactobionate. In this study the UW gluconate solution was used for the continuous hypothermic machine perfusion of dog livers for 72 hr. Dog livers were continuously perfused at 5 degrees C through the portal vein at a pressure of 16-18 mm Hg and transplanted. Seven of 8 dogs survived for 7 or more days following orthotopic transplantation. The livers functioned as well as those preserved for 48 hr by cold storage in the UW solution as indicated by various liver-function tests. Successful machine perfusion was only achieved when the perfusate contained a high concentration of potassium (125 mM) but not with a high concentration of sodium (125 mM). This study demonstrates the feasibility of machine-perfusion preservation of the liver that yields longer preservation of equal quality compared to simple cold storage. For the development of truly long-term preservation (5 or more days) and better quality short-term preservation, machine perfusion may be the method of choice.

THE ROLE OF COLD ISCHEMIA IN A PROVINCIAL ORGAN-SHARING PROGRAM IN THE CYCLOSPORINE ERA

Prolonged cold ischemia has been associated with impaired early cadaver renal allograft function. The role of CsA in potentiating these effects is not well understood, but CsA has been implicated in promoting delayed graft function and potentiating renal ischemic injury. In order to establish whether CsA is safely tolerated by kidneys subjected to protracted cold ischemia, we examined patient and graft outcome in a series of 1081 patients receiving cadaver-kidney transplants over an 8-year period (1981-1988). All patients received a standard immunosuppressive regimen that included CsA. Overall actuarial 1-year patient and graft survival rates were 96% and 80%, respectively. Renal preservation was achieved either by pulsatile perfusion (n = 261, 24%) or simple cold storage (n = 820, 76%). Results were analyzed according to total cold ischemic time as follows: 0-23 hr (n = 512; range, 0-23.9 hr); 24-35 hr (n = 380; range, 24.0-35.9 hr); 36-47 hr (n = 161; range, 36.0-47.7 hr); greater than or equal to 48 hr (n = 28; range, 48.0-70.6 hr). These groups did not differ significantly in recipient age, sex, incidence of diabetes, number of pretransplant blood transfusions, level of presensitization, or HLA match. There were no differences in overall actuarial 1-year patient or graft survival rates, incidence of rejection, or renal function at 1 year. There was a higher incidence of impaired early graft function for kidneys preserved greater than or equal to 48 hr, but eventual graft outcome, including serum creatinine at 1 year, was unchanged. Delayed introduction of CsA resulted in improved 1-year graft survival (84.4% vs. 74.7%, P less than 0.05) compared to CsA treatment begun at the time of transplantation ("initial CsA"). This improvement was present regardless of total cold ischemia time. The incidence of permanent graft nonfunction, which has been previously reported to increase with CsA therapy, was influenced by the timing of CsA therapy (initial: 12%; delayed: 3%, P less than 0.05) but was not affected by duration of cold ischemia. Thus, safe preservation of cadaver kidneys for up to 70 h can be achieved by standard techniques even when CsA is incorporated into the immunosuppressive regimen. The most important determinants of graft survival in these patients are the timing of CsA therapy and the presence of early graft function, not the duration of renal preservation.

Hypothermic preservation of hepatocytes: I. Role of cell swelling

Hepatocytes from isolated rat livers were hypothermically incubated (5 °C) in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). The incubation solution was either a tissue culture medium (L-15), an organ preservation perfusate (UW gluconate), or a simple cold-storage solution used for organ preservation (UW lactobionate). Hepatocyte viability was assessed from the release of lactate dehydrogenase (LDH) into the incubation medium. Cell swelling (due to the uptake of water) was also measured. Within 24 hr, hepatocytes hypothermically stored in each of the three incubation solutions became swollen (30 to 40% water gain) and lost a significant amount of LDH (as much as 60%). The addition of polyethylene glycol (PEG; relative molecular mass 8000; 5 g%) to the solutions suppressed cell swelling and allowed the incubated hepatocytes to remain relatively well preserved (30% LDH release) for as long as 120 hr. Adding either dextran (relative molecular mass 10,000 to 78,000; 5 g%) or saccharides (100 mmol/liter) instead of PEG neither prevented cell swelling nor prevented the cells from dying. The results of this study suggest (i) there is a direct correlation ( r = 0.873) between hypothermia-induced cell swelling and cell death (i.e., the suppression of cell swelling prevents cell death); (ii) the mechanism by which PEG prevents cell swelling (and thus maintains cell viability) is not related to the osmotic or oncotic properties of the molecule but instead is apparently related to some unknown interaction between PEG and the cell, an interaction that provides stability during hypothermie incubation; and (iii) hypothermia-induced cell swelling must be prevented if isolated hepatocytes are to be used as a model for studying the mechanism by which cell damage occurs during hypothermic organ preservation. By eliminating cell death due to cell swelling, the biochemical mechanisms of cell death can be studied.

Seventy-two-hour preservation of the canine pancreas by the two-layer (Euro-Collins' solution/perfluorochemical) cold storage method.

A 72-hr preservation of canine pancreas by a 2-layer (Euro-Collins'/Perfluorochemical) cold storage method was tested in the canine model of segmental pancreas autotransplantation. The functional recovery of the grafts by this method (group 1) was determined by daily fasting blood glucose concentration and intravenous glucose tolerance test at 2 weeks after autotransplantation and compared with simple cold storage with Euro-Collins' solution (group 2) and control (no preservation) (group 3). Maintenance of normoglycemia for at least 5 days after transplantation was considered a successful preservation. The functional success rates after 72-hr preservation were 100%, 0%, and 100% for groups 1, 2, and 3, respectively. The mean K values of group 1 after 72-hr preservation was 1.78 +/- 0.42 compared with 2.05 +/- 0.32 for group 3 at 2 weeks after transplantation. Biopsies of grafts of group 2 after 72-hr preservation showed remarkable autolytic changes in exocrine and endocrine tissues. In contrast, biopsies of grafts of group 1 after 72-hr preservation showed almost normal architecture in both tissues. In addition, biopsies of 72-hr preserved grafts of group 1 at 4 weeks after after autotransplantation showed almost normal pancreas architecture with minimal fibrotic changes in the exocrine tissue. This study demonstrated the possibility of 72-hr preservation of the pancreas for transplantation.

Experimental pancreatic preservation prior to islet isolation and transplantation

Preservation of the Lewis rat pancreas prior to islet isolation was accomplished by initial intraductal distension with the University of Wisconsin (UW) hydroxyethyl starch-lactobionate solution to which collagenase had been added, followed by simple cold storage at 4°C for 0, 3, 12, 24, and 48 hr ( n = 16–21 at each interval). The pancreases were then processed by digestion and mechanical dispersion to produce free islets of Langerhans. The mean islet yields (± standard errors of the means) were controls = 819 ± 58 ( n = 21), 3 hr = 867 ± 51 ( n = 20), 12 hr = 770 ± 71 ( n = 16), 24 hr = 805 ± 62 ( n = 18), and 48 hr = 722 ± 55 ( n = 16). None of these means differed significantly. The islets from pairs of donor pancreases (mean dose of islets = 1586 ± 72) were transplanted intraportally into single isogeneic recipients with streptozotocin-induced diabetes (plasma glucose > 400). The preservation interval directly influenced the outcome of these islet isografts in the following manner: (i) Rates of functional success (nonfasting glucose < 200 mg/dl) were 100% after storage times of 0 hr ( n = 10), 3 hr ( n = 8), and 12 hr ( n = 8); 86% with a storage time of 24 hr ( n = 7); and 0% after 48 hr ( n = 8). (ii) Return of euglycemia was increasingly delayed with increasing preservation intervals. Controls were all euglycemic after 1 day, whereas the mean times (±SEM) to euglycemia in the preservation groups were 3 hr = 4.4 ± 1.8 days, 12 hr = 6.0 ± 2.5 days, and 24 hr = 7.0 ± 2.9 days. (iii) Intravenous glucose tolerance was impaired in those animals receiving islets from pancreases stored for > 12 hr, when compared with animals receiving fresh islets. Mean K values (1–15 min) were controls = −4.07 ± 0.36, 3 hr = − 4.81 ± 0.39, 12 hr = − 5.38 ± 0.87, and 24 hr = − 2.56 ± 0.55. The mean value in the 24-hr group was significantly different from that in the controls ( P < 0.045) and other preservation groups ( P < 0.027). Thus, while 24-hr pancreatic storage prior to islet isolation and transplantation is feasible in this model, there is evidence for both reversible and irreversible injury to islets that is related to the length of the preservation interval. This has implications for attempts at human islet transplantation when cadaveric pancreases are used as the source of islets.

The beneficial effect of intermediate normothermic perfusion during cold storage of ischemically injured kidneys. A study of renal nucleotide homeostasis during hypothermia in the dog.

The effect of simple cold storage on ice with or without preceding warm ischemic injury on the energy metabolism and posttransplant viability of canine kidneys was examined in the present study. In addition, we investigated the possible beneficial effect of an intermediate normothermic perfusion half-way through the storage period on the preservation of ischemically injured kidneys. Thirty mongrel dogs were allocated to 5 experimental groups. In groups I and II kidneys were simply stored on ice for 24 and 48 hr, respectively. In groups III and IV kidneys were additionally subjected to 30 min warm ischemia before storage. In group 5 kidneys were treated as in group IV, but halfway through the storage period an intermediate normothermic ex-vivo perfusion was performed. The effect of these procedures on renal viability was tested by autologous reimplantation of the kidneys. During implantation the contralateral kidney was immediately removed. In group I all animals survived, whereas in group IV none of the animals survived. In groups II, III, and V, 2 of 6, 1 of 6, and 3 of 6 animals survived, respectively. The relationship, if any, between poststorage renal viability and the tissue levels of adenine nucleotides, guanine nucleotides, IMP, and purine degradation products was assessed by measuring the content of these metabolites in tissue specimen of the renal cortex, on which biopsies were done at various intervals during the experimental procedures. After an initial drop of about 30% in the content of adenine and guanine nucleotides and an increase in IMP, these values remained constant during 48 hr of cold storage. In contrast to kidneys stored for 24 hr, reimplantation of kidneys stored for 48 hr resulted in a significant decrease of adenine nucleotides following 60 min of in vivo reperfusion. Warm ischemia for 30 min prior to cold storage lead to lower initial nucleotide levels at the start of the storage period. During the first 24 hr nucleotide levels did not change, but a further decrease was observed during the following 24 hr of storage. Reimplantation after 24 hr of storage resulted in an additional decrease in the content of nucleotides. This poststorage decrease was absent after 48 hr of cold storage. Intermediate normothermic perfusion halfway through the storage for 48 hr significantly prevented the drop in the nucleotide content observed during the last 24 hr of storage in the corresponding control group. This nucleotide-sparing effect did not increase the level of nucleotides at the end of 60 min of reperfusion following reimplantation.(ABSTRACT TRUNCATED AT 400 WORDS)

Clinical application of kidney perfusion preservation machine-An immediate functional recovery of cadaveric kidney grafts by hypothermic perfusion preservation (HPP) compared with simple cold storage (SCS)

Clinical application of kidney perfusion preservation machine-An immediate functional recovery of cadaveric kidney grafts by hypothermic perfusion preservation (HPP) compared with simple cold storage (SCS)

An analysis of the components in UW solution using the isolated perfused rabbit liver.

The isolated perfused rabbit liver model has been used to determine the essential components of the UW solution for hepatic preservation by simple cold storage. Livers were stored on ice for 48 hr after initial flushing with the solution being tested, and then reperfused at 38 degrees C in an isolated perfusion circuit; bile flow and enzyme (SGOT, SGPT, and LDH) release during a 2-hr period were recorded. All solutions tested contained phosphate (25 mM) as a buffer and magnesium sulfate (5 mM). Sodium can be substituted for potassium without adverse effects. Lactobionate, raffinose and glutathione cannot be omitted; all other components can be eliminated without altering the effectiveness of the solution in this model.

Improvement of renal preservation by verapamil with 24-hour cold perfusion in the isolated rat kidney.

There is substantial evidence that increased cellular calcium may activate processes that lead to cellular injury and death, and calcium entry blockers (CEB) have been shown to protect against renal ischemic injury. This approach has been used experimentally to enhance kidney preservation during both warm and cold ischemia. In the present study, the effect of the CEB verapamil on kidney function after 24 hr of hypothermic (4-7 degrees C) perfusion was examined and compared with simple cold storage with Eurocollins' solution (4 hr), 4 or 24 hr cold perfusion, without the addition of verapamil. The cold perfusion media consisted of 3% albumin in phosphate-free Krebs-Henseleit saline supplemented with 5 mM glucose. Cold perfusion was performed at 40 mmHg perfusion pressure with either 0 (C) or 5 microM verapamil (V) added to the cold perfusion media. Renal functional parameters of plasma flow (RPF), inulin clearance (Cin), fractional (FRNa+) and net sodium reabsorption (TNa+) were assessed during 60 min of reperfusion at 37 degrees C using 6.7% albumin in Krebs-Henseleit saline supplemented with glucose, inulin, and 20 amino acids. There was no increase in RPF with V (33 +/- 1 vs. 32 +/- 2 ml/min/g,NS) but Cin was significantly higher (271 +/- 30 vs. 168 +/- 20 microliter/min/g P less than 0.01) with V. Preservation of tubular function by V was demonstrated by an increase in FRNa+ (84 +/- 5 vs. 57 +/- 8%, P less than .01), TNa+ (32 +/- 6 vs. 15 +/- 3 mumol/min/g, P less than .01) and renal adenosine triphosphate (ATP) concentration (8.0 +/- 5 vs. 4.7 +/- 1.0 mumol/g dry tissue, P less than .01). Thus, V appears not only to enhance kidney preservation with warm and cold ischemia but also improves renal function, as assessed by glomerular filtration rate (GFR) tubular function, and tissue ATP concentration with 24-hr cold perfusion.

IMPROVED IMMEDIATE FUNCTION OF RENAL ALLOGRAFTS WITH BELZER PERFUSATE1 Pressented at the 6th Annual Meeting of the American Society of Transplant Physicians, May 1987, Chicago, IL

The characteristics of 254 cadaveric kidneys were evaluated and the incidence of immediate function identified. The Belzer perfusate was used primarily (n = 140) and secondarily (n = 14) in combination with pulsatile machine perfusion. These two groups were compared with a previous group of kidneys machine-perfused with silica gel (cryoprecipitated human plasma). The incidence of immediate function of the group primarily perfused with Belzer perfusate was statistically significantly improved over that of the silica gel. The secondarily perfused Belzer group, "imported" kidneys previously preserved with simple cold storage, had notably longer periods of preservation and higher resistances on the machine. However, 100% of this group functioned immediately. Other findings in this study show that the Belzer perfusate allows for improved parenchymal function posttransplant, as noted by a more rapid clearance of serum creatinine posttransplant. When comparing the immediate function group with those suffering early dysfunction, there is a statistically significant increased resistance on the machine in the latter group. This allows for prediction of immediate function based on perfusion characteristics of the kidney. The Belzer perfusate, composed of metabolic substrates for high-energy phosphate production, improves the incidence of immediate function in machine-perfused kidneys, as well as improved qualitative function posttransplant. It also is effective as a "rescue" mechanism in previously simple cold-stored (ATP-depleted) kidneys.