Evidence that graft survival is not related to parenchymal cell viability in rat liver transplantation. The importance of nonparenchymal cells

Injury to parenchymal and nonparenchymal cells of livers stored in cold Euro-Collins solution was assessed following reperfusion and compared with graft survival following orthotopic rat liver transplantation. Parenchymal cells maintained their viability nearly completely after up to 24 hr of cold storage as assessed by trypan blue exclusion (97% of cells) and LDH release (4% of total) from livers reperfused for 20 min following storage. Furthermore, hepatic glycolysis (rates of lactate plus pyruvate production), oxygen uptake and NADH redox state (lactate:pyruvate ratio) were in the normal range at all time points studied up to 24 hr of cold storage. In contrast, nonparenchymal cells lost viability as assessed from trypan blue staining beginning after 8 hr of storage: 40% were nonviable after 24 hr of storage. Since injury to nonparenchymal cells occurs only upon reperfusion, oxygen radicals may be involved. Accordingly, xanthine and hypoxanthine, substrates for oxygen radical formation, were measured in perfusate upon reperfusion. Both purines accumulated (up to 80 microM) with time of storage and were washed out rapidly (less than 10 min) upon reperfusion. Although parenchymal cell function was in the normal range in livers stored in the cold for 24 hr, liver grafts stored for 6 hr and longer in Euro-Collins solution could not be transplanted successfully. Thus, we conclude that viability of parenchymal cells in liver grafts prior to transplantation is a poor parameter to predict the outcome of transplantation. Therefore, assessment of parenchymal cell energy state (e.g., with 31P NMR and other methods) most likely will not predict survival reliably. On the other hand, nonparenchymal cells lose their viability significantly earlier following storage and reperfusion. These data suggest that preservation of nonparenchymal cell viability is critical for successful liver transplantation.     

The use of myocytes as a model for developing successful heart preservation solutions

The development of a successful method to preserve the heart for relatively long periods (24-48 hr) requires demonstrating successful orthotopic transplantation and long-term survival after preservation. There are, however, multiple variables that may affect the quality of heart preservation, and it is nearly impossible to systematically study all the variables in this complicated model. One model that may be useful to study how preservation parameters affect heart cell preservation is the isolated myocyte preparation. In this study myocytes were isolated from the rabbit heart and the effects of up to 24 hr cold storage on viability measured to determine if this would be a suitable preservation model. Myocytes were stored in various preservation solutions including; EuroCollins (EC), two cardioplegic solutions (Stanford [ST] and Bretschneider solution [HTK]) and the University of Wisconsin solution (UW) with or without the addition of polyethylene glycol. The viability of myocytes was judged by measuring the effects of preservation and rewarming after preservation on cellular morphology (percent rod-shaped cells), ATP concentration, and LDH release. Myocytes preserved in the cardioplegic solutions were least well preserved after 12 and 24 hr storage, as judged by the loss of rod-shaped morphology and lower ATP concentration. Preservation in EC resulted in a decrease in the percent rod-shaped cells after 12 hr and 24 hr storage that was greater than obtained in the UW solutions. The best preservation of myocyte morphology and highest content of ATP was obtained in myocytes stored in the UW solutions, especially those containing PEG. The myocyte model of heart preservation shows a loss of cell integrity that is related to the preservation solution (HTK greater than ST greater than EC greater than UW-PEG) and these results are similar to what has been shown in the past with other models of heart preservation. Thus the myocyte model appears to be a useful method to test how many preservation solutions and preservation variables affect heart cell metabolism. In the future, results from these types of studies may find use in developing improved heart preservation solutions for testing in the orthotopic transplant model.     

Biphasic mechanism for hypothermic induced loss of protein synthesis in hepatocytes.

BACKGROUND: A complication in liver transplantation is increased clotting times due to inhibition of protein synthesis resulting from prolonged hypothermic preservation. Protein synthesis is also blocked in cold preserved hepatocytes. In this study, the mechanism of inhibition of protein synthesis in cold preserved hepatocytes was investigated. METHODS: Hepatocytes prepared from rat liver were cold preserved in University of Wisconsin solution for 4, 24, and 48 hr. Protein synthesis was measured as incorporation of radiolabeled leucine into acid precipitable proteins. Hepatocytes were treated with antioxidants (dithiothreitol, trolox or deferoxamine, nitric oxide synthase inhibitor (N(G)-monomethyl-L-arginine monoacetate), steroids (dexamethasone or methylprednisolone), methods to keep adenosine triphosphate high (aerobic storage), and cytoskeletal disrupting agents (cytochalasin D or colchicine). RESULTS: There was a 26% decrease in protein synthesis after only 4 hr of cold storage and a further 25% decrease at 24 hr. Antioxidants, elevated adenosine triphosphate, and N(G)-monomethyl-L-arginine monoacetate did not affect the rate of loss of protein synthesis. Protein synthesis was not due to inhibition of amino acid transport or lack of amino acids in the storage medium. Steroid pretreatment of hepatocytes had no effect on the loss of protein synthesis occurring in the first 4 hr of storage but did suppress the loss occurring during the next 44 hr of storage. Cytoskeletal disrupting agents, added to freshly isolated cells, inhibited protein synthesis. CONCLUSION: The mechanism of loss of protein synthesis in cold preserved liver cells is not mediated by: (1) oxygen free radical generation or improved by antioxidant therapy, (2) nitric oxide generation in hepatocytes, (3) an adenosine triphosphate-sensitive destruction of cell viability, and (4) decreased permeability of amino acids or loss of amino acids from the cells. Loss of protein synthesis due to hypothermic storage appears biphasic. The first phase, occurring within 4 hr of storage, may be the result of the effects of hypothermia on the cell cytoskeletal system and may be untreatable. The second phase, which occurs during the next 24 to 48 hr is sensitive to steroid pretreatment. This phase may be amenable to improved preservation methodology. Improved preservation of the liver may require the use of steroids to conserve protein synthetic capabilities.     

Improvement of endothelial cell viability at 4 degrees C by addition of lazaroid U74500A to preservation solutions.

Lazaroids are potent inhibitors of lipid peroxidation. Endothelial cell damage has been shown to occur during cold storage preservation of lung and liver. This study examines the effects on endothelial cell viability of the addition of four lazaroids, U74006F, U78518F, U74500A, or U75412E to preservation solutions. Human umbilical vein endothelial cell cultures were stored at 4 degrees C for 48 and 96 hr in EuroCollins or 5% polyethylene glycol in buffered saline (PEG). U78518F, U74500A, U74006F, U75412E, or dexamethasone (each 50 microM) was added to EC (n = 32) or PEG (n = 32) and compared with control solutions of EC or PEG alone. Endothelial cell viability was determined by measuring cellular reduction of 3-[4,5-dimethylthiazol-2-yl]-2,3-diphenyltetrazolium bromide to a purple formazan dye. The reduction occurs only in viable cells and requires mitochondrial dehydrogenase activity. Results were quantified by measuring dye absorbance (Ab) with a micro-ELISA spectrophotometer. Absorbance values were compared by ANOVA and reported as mean values +/- standard deviation. Addition of U74500A to EC (Ab = 0.474 +/- 0.055) and PEG (Ab = 0.462 +/- .005) improved viability at 48 hr when compared with EC (Ab = 0.289 +/- 0.069) and PEG (Ab = 0.287 +/- 0.052) alone (P less than 0.05). At 96 hr, addition of U74500A resulted in improved viability in both EC (Ab = 0.377 +/- 0.068) and PEG (Ab = 0.195 +/- 0.09) or PEG alone (Ab = 0.212 +/- 0.1) (P less than 0.05). Other lazaroids tested were also effective in improving cellular viability, but to a lesser degree than U74500A. This study demonstrates that the addition of lazaroids to organ preservation solutions improves endothelial cell viability.     

Viability of Cryopreserved Parathyroid Tissue: When Is Continued Storage versus Disposal Indicated?

Background Parathyroid cryopreservation is used for potential autografting in patients who are rendered hypocalcemic following surgery. Cryopreservation employs multiple resources and carries a significant cost for processing and storage of tissue. Importantly, the length of time that parathyroid tissue remains functional after cryopreservation is not known. The goal of our study was to assess ex-vivo viability of parathyroid tissue in relation to the length of time in storage. We sought to define the appropriate time frame for tissue utilization and disposal to assist with long-term surgical planning. Methods From 1991 to 2006, 501 parathyroid specimens from 149 patients were cryopreserved at −80°C according to standardized techniques. A single trained technician assessed viability, using a hemacytometer to count viable (clear cell) and nonviable (blue cell) tissue. Univariate analysis was performed to correlate length of preservation, diagnosis with viability. Results: We evaluated 106 random parathyroid specimens. Samples were divided into two groups: those stored > 24 months and those stored ≤ 24 months. Eleven (10%) of 106 specimens were deemed viable. Length of storage time was associated with ex-vivo tissue viability. Viability of samples cryopreserved ≤ 24 months was 71% (10/14) versus 1% (1/92) of those specimens stored > 24 months (p < 0.001). Conclusions Viability of cryopreserved parathyroid cells is associated with duration of storage. Parathyroids preserved for greater than 24 months are unlikely to be viable. It seems reasonable to limit parathyroid cryopreservation to 24 months when frozen at −80°C. Further studies are needed to optimize the process of cryopreservation to enhance cell viability.     

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)     

Cold-preserved rat liver viability testing by proton nuclear magnetic resonance relaxometry.

The results of a series of experiments in the cold-preserved rat liver, applying a newly developed method of pretransplant viability testing, are described. The livers were stored either under simple hypothermic conditions (KHB) or in EC, HTK, or UW preservation solution. Livers were stored up to 48 hr and reperfused after a period of hypothermic storage of 1, 7, 14, or 21 hr. In a parallel series of experiments, with livers stored under identical conditions, repeated proton relaxometry measurements (0, 1, 7, 14, 24, 32, 48 hr) were undertaken; and ATP, ADP, AMP (Atkinson's energy charge), and water content of livers, as well as pH of storage solution, were estimated. Based on a strong correlation between proton spin-spin relaxation time T2 and tissue water content (edema), this new method may be useful to estimate the amount of cell swelling during hypothermic storage from a surgical biopsy of about 200 mg within a few minutes. There was, however, no significant correlation found between energy charge and/or pH and water content, T2, or bile flow. Our method could be useful as a rapid test method in experimental cold liver storage models and may be of interest in human liver transplantation as a viability indicator in combination with other parameters.     

Effect of argon laser and Photofrin II on murine neuroblastoma cells.

Laser-induced fluorescence (LIF) of photosensitizers is used to detect cancer. The effect of argon laser light with an average irradiance of 31 mW cm-2 and Photofrin II (Dihematoporphyrin ether, DHE) at concentrations of 1.0 and 5.0 micrograms ml-1 on C1300 murine neuroblastoma cells (MNB, NB41A3) in vitro was investigated. Growth curves and cell viability (trypan blue dye exclusion) were determined at 1, 24, 96, and 144 hr post-irradiation. Light doses of 1.8 and 9.0 J cm-2 combined with 5.0 micrograms DHE ml-1 decreased both cell numbers and viability, immediately and up to 144 hr postirradiation. Argon laser light alone at a fluence of 9.0 J cm-2 caused reversible injury to the cells. This in vitro study shows that both low energy argon laser light and low dose DHE are cytocidal to C1300 MNB cells. LIF promises to aid in the detection and destruction of neuroblastoma. Surgeons should be aware that tissue irreversible damage is likely to occur when performing LIF detection of neuroblastoma. The doses of laser light and of Photofrin II found to be toxic to neuroblastoma cells in culture may provide guidelines for photodynamic therapy ablation of neuroblastoma clinically.     

Hypothermic Storage of Periportal and Perivenous Rat Hepatocytes

High yields of intact parenchymal cells are produced by the two-step Digitonin-collagenase perfusion of whole liver, and it has gained wide acceptance for biochemical and cellular analyses of zonal hepatocytes. The development reached by this methodology is in contrast to the time-limited use of the isolated cells unless those other methods, such as primary cultures, are employed. An alternative option to have cells ready to be used for several days, is the cold storage in University of Wisconsin solution as a preservation solution. This procedure is easy, not too expensive, and does not require specialized equipment. We study the competence of this system to maintain liver cells: mixed or total cells and cell-enriched fractions. We affirm viability of hepatocytes during hypothermic storage (UW-96h-4°C) by Trypan Blue exclusion, the capacity to retain cytoplasmic enzymes, metabolic competence to maintain total Glutathione content, and immunocytochemistry (gene detection). After 96 h of cold storage, mixed cells and cell-enriched fractions, were submitted to normothermic incubation (120 min, 37°C) and we check Trypan Blue exclusion, cytoplasmic enzyme release, and the capacity of cell populations to synthesize urea. The results show that it is possible to use, after several days of storage, mixed liver cells and cell-enriched fractions in metabolic and gene expression studies. This procedure allows us to reduce the number of experimental animals needed, to save experimental time and costs, and to facilitate further studies in vitro about the basis and consequences of metabolic heterogeneity of the liver cell plate.     

Blood loss and factors affecting pulmonary antibacterial defenses

Since impaired intrapulmonary bacterial inactivation of staphylococcus aureus has been described in rats during the first 15 hr after blood loss, the effect of phlebotomy on several factors known to influence host responses to bacteria in the lung were studied.Significant hypotension was present during the first hour after phlebotomy, but arterial pressure was normal during the next 14 hr. Respiratory alkalosis was present at one, but not at 4 or 15 hr after blood loss. Fractional lung water content was increased at both 4 and 15 hr. Alveolar bubble stability was depressed at both 4 and 15 hr. Phlebotomy had no effect on the alveolar macrophage when it was studied in vitro. Sham and experimental rats had similar number, cell viability, oxygen consumption and enzyme content of recovered macrophages at 4 and 15 hr.Since in vitro studies have demonstrated the need of normal surfactant fraction for maximal bactericidal activity of the alveolar macrophage, the abnormal surface lining material with blood loss may have contributed to impaired in vivo host defenses. Study of humoral and immunological influences on the alveolar macrophage after blood loss is needed to further evaluate mechanisms of intrapulmonary bacterial inactivation.     

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.     

Addition of a water-soluble alpha-tocopherol analogue to University of Wisconsin solution improves endothelial viability and decreases lung reperfusion injury.

BACKGROUND: Reperfusion injury following lung preservation has been associated with free radical formation and subsequent endothelial cell damage. Trolox is a water-soluble analogue of the free radical scavenger alpha-tocopherol. We hypothesized that addition of this form of vitamin E to University of Wisconsin (UW) solution would decrease reperfusion injury and improve lung function after cold ischemic preservation. MATERIALS AND METHODS: Bovine aortic endothelial cells were cultured and stored at 4 degrees C for 12, 24, and 48 h in UW or UW + Trolox (UWT). Endothelial cell viability after storage was assessed by dimethylthiazole tetrazolium cytotoxicity assay. An isolated rat perfused lung (IPL) model was used and lungs were flushed with the respective solutions with cold storage times of 6 and 12 h. Following storage, the lungs were reperfused with fresh blood and lung function was assessed by blood gas analysis, alveolar-arterial gradient, and compliance. RESULTS: There was no difference in endothelial cell viability between UW and UWT after 12 or 24 h; however, UWT had higher endothelial cell viability than UW with 48 h of cold ischemic storage. Using the IPL model, the pO2 was higher with UWT than UW after 6 and 12 h of cold ischemia. The alveolar-arterial oxygen difference was significantly lower for UWT versus UW at 6 h. UWT provided increased compliance at 6 and 12 h of ischemia. CONCLUSIONS: The addition of a water-soluble vitamin E analogue to UW solution resulted in increased endothelial cell viability after prolonged storage and improved whole lung preservation in the postreperfusion period as evidenced by higher oxygenation and increased compliance. These results are clinically relevant as the lung is extremely sensitive to reperfusion injury and UW solution is being increasingly used in lung transplantation and remains the predominant solution in abdominal organ transplantation.     

The integrity of tubular cell function after preservation in Collins' or Sacks' solution.

Collins' solution is an excellent medium for kidney preservation by simple cold storage before transplantation. Efforts by Sacks et al. to improve this technique by modifying the composition seemed promising. A comparison between ability of these two media to preserve the tubular cell was attempted. The viability of the tubular cells was evaluated by measuring the 125 I Hippuran uptake in cortex slices taken from kidneys preserved for 24 and 48 hr in the respective solutions. Kidneys exposed to as well as kidneys not exposed to warm ischaemia were used. Collins' solution was found to be superior in protecting the cell function. Nonetheless intracellular oedema during preservation was greater after preservation in this solution as compared with cells preserved in Sacks' solution.     

Superiority of the two-layer method before islet isolation confirmed by in vivo viability assessment

BACKGROUND: Although the outcome of islet transplantation has improved, there remains a major obstacle in isolating viable islets from prolonged preserved pancreas. We previously reported that the two-layer cold storage method (TLM) improved the yield and in vitro function. In this study, we performed in vivo accurate functional analyses of islets from TLM-preserved pancreas and investigated pancreatic duct cell viability, which may critically affect islet isolation. METHODS: Rat islets isolated from fresh pancreas (group 1), after preservation in the University of Wisconsin (UW) solution (group 2) or by the TLM (group 3), were examined by assessing islet yields, stimulation indices, cure rates after transplantation to diabetic nude mice, and trypan blue uptake of pancreatic duct cells. RESULTS: TLM significantly improved the islet yield compared with UW cold storage. The cure rates after transplantation were 100%, 0%, and 80% for groups 1, 2, and 3, respectively. This indicates that islet viability was well maintained even after 24 hr of TLM preservation. The percentages of nonviable duct cells were 4.1%+/-1.9%, 48.3%+/-8.0%, and 26.1%+/-21.4% in groups 1, 2, and 3, respectively, showing that the TLM was superior to UW as seen by this duct cell viability assessment. CONCLUSIONS: The TLM used for pancreas preservation before islet isolation results in excellent islet function in addition to improved islet yield comparable to freshly isolated islets. The underlying mechanism may be duct cell viability maintained during TLM preservation. Therefore the TLM is an excellent preservation technique for isolating sufficient numbers of highly viable islets.     

The mechanism of action of the two-layer (Euro-Collins' solution/perfluorochemical) cold-storage method in canine pancreas preservation--the effect of 2,4 dinitrophenol on graft viability and adenosine triphosphate tissue concentration.

Tissue concentrations of adenosine triphosphate have been previously associated with successful pancreas preservation using the two-layer cold storage method in a canine autotransplantation model. To clarify the role of ATP vs. oxygenation per se, we used 2,4 dinitrophenol, an uncoupler of mitochondrial oxidative phosphorylation. DNP caused no toxicity in pancreas grafts preserved for 24 hr in Euro-Collins' solution or 48 hr in University of Wisconsin solution. Tissue concentration of ATP and viability of pancreas grafts, defined as maintenance of normoglycemia for 5 days following transplantation, were compared among six groups after a preservation interval of 24 or 48 hr. After 24 hr all grafts were viable, whether preserved using simple cold storage in EC (group 1a), two-layer (EC/perfluorochemical [PFC]) method (group 2a), or two-layer (EC+DNP/PFC) method (group 3a); respective graft survival was 4/4 (100%), 5/5 (100%), and 4/5 (80%); one of five dogs in group 3a died of a cause unrelated to the pancreas. ATP levels were higher in group 2a compared with group 1a (7.47 +/- 0.47 vs. 1.41 +/- 0.53 mumol/g dry weight, P less than 0.01) and lower in group 3a compared with group 2a (1.25 +/- 0.37 vs. 7.47 +/- 0.47, P less than 0.01). After 24 hr, we observed no difference in viability despite ATP concentration differences. However after 48 hr preservation, graft viability varied among the groups: 0/4 (0%), 4/4 (100%), and 0/3 (0%) in groups 1b, 2b, and 3b, respectively. ATP tissue concentration was again higher in group 2b after two-layer (EC/PFC) method preservation (7.91 +/- 1.21 vs. 1.21 +/- 0.31 mumol/g dry weight, P less than 0.01) compared with EC preservation (group 1b). DNP again caused a significant decrease in tissue ATP in group 3b (0.61 +/- 0.07 vs. 7.91 +/- 1.21, P less than 0.01). The two-layer (EC/PFC) method clearly protected pancreas viability, and inhibition of ATP production using DNP caused loss of viability in this model. We conclude that oxygenation of the pancreas during preservation by the two-layer method allows continued ATP production within the graft. Metabolic processes vital to cellular integrity can be maintained, which produces an extended period of preserved pancreatic viability.     

Advantages of normothermic perfusion over cold storage in liver preservation

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

Development of an optimal rechargeable cardiac pacemaker

While many rechargeable pacers, including a commercial unit have been developed using nickel-cadmium cells, they are in general limited by low cell capacity, short life without recharging, the need for frequent recharging, indirect monitoring of battery voltage by stimulation rate changes and gradually falling cell capacity with each cycle at body temperature.We have developed a low-drain rechargeable pacemaker, hermetically sealed in a seamless container, that will stimulate continuously for 4 yr without recharging. Our battery is a silver modified 1000 mA hr rechargeable version of the mercury-zinc cell proven by years of use in over 95% of pacers used clinically to date. It has functioned for over 6 yr in real time tests and in 120 accelerated tests has always simulated over 50 yr of pacing. Our pacer is small and thin, with less than one-half the displacement of current nonrechargeable units. Electromagnetic recharging energy is painlessly transmitted through the intact skin by a tuned coil, using a unique system unaffected by variations in implant depth. A telemetry oscillator permits noninvasive monitoring and telephone transmission of cell voltage, stimulation rate, implant depth, and optimal recharging coil location. Six animal and 3 clinical implants continue perfect function after up to 2.5 and 1 yr, respectively. A 6 yr life is proven and a greater than 20 yr life is anticipated.     

Reconstituted thiopentone retains its alkalinity without bacterial contamination for up to four weeks

The manufacturers of thiopentone recommend that after reconstitution, it should be kept only for 24 hr to reduce the risk of contamination. However, there are no studies to support this practice and compliance with this recommendation has economic implications. The reasons for discarding a reconstituted bottle of thiopentone are related to concerns about chemical and physical (pH) stability, contamination with infectious agents, and contamination with cellular material. We studied the incidence of bacterial contamination and pH stability of thiopentone in clinical use, as well as the pH stability of thiopentone not in clinical use, and surveyed the eight hospitals affiliated with the University of British Columbia to determine their protocols for thiopental preparation and storage. Cost comparisons were made between our current practice of discarding thiopentone when depleted and the practice of routinely discarding it 24 hr after reconstitution. Samples of thiopentone in clinical use were cultured daily and the pH was measured. The bottles had been in clinical use from 1 to 25 days (mean 4.23 ± 4.32 SD). Of 106 samples there were no positive bacteriological cultures and there were only minor changes in pH. The telephone survey of the eight hospitals revealed that only one had a policy to discard thiopentone after 24 hr. Cost comparisons indicate that discarding thiopentone 24 hr after reconstitution would result in increased cost. In conclusion, reconstituted thiopentone retains its alkalinity for up to four weeks, and has an acceptably low risk of bacterial contamination for periods beyond 24 hr, therefore thiopentone need not be discarded after 24 hr.     

Apoptosis versus necrosis during cold storage and rewarming of human renal proximal tubular cells

BACKGROUND: A recent clinical study demonstrated that in renal allografts preserved in the cold apoptosis occurred soon after reperfusion. The mode of cell death during cold storage is generally considered necrotic. Whether apoptosis occurs as a part of cold storage is uncertain. The objective was to determine in human renal tubular cells whether apoptosis is specific for rewarming or it also occurs during cold storage and whether it could be modified. METHODS AND RESULTS: Cold storage (4 degrees C) of primary human renal proximal tubular epithelial (RPTE) in University of Wisconsin (UW) solution up to 48 hr caused a time-dependent increase in cell death measured by lactic dehydrogenase (LDH) release and vital dye exclusion methods. Transmission electron microscopy (TEM) demonstrated that cell death in the cold was necrotic, involving considerable mitochondrial disruption, and was not apoptotic. The TUNEL assay that provides a specific, quantitative measure for apoptosis showed no increase in TUNEL-positivity during flow cytometry of cells stored in cold: 37 degrees C, 0.23+/-0.14%; 24 hr cold, 0.23+/-0.1%; 48 hr cold, 1.79+/-0.58%. Annexin-V staining, a sensitive method for detecting early apoptosis, similarly showed no increase in positively stained cells during cold storage. Addition of antioxidants 2-methyl aminochroman and deferoxamine to UW solution inhibited necrotic cell death and preserved mitochondrial structure. In contrast to cold storage alone, rewarming (37 degrees C for 24 hr) of cold stored cells, however, resulted in significant apoptosis (TUNEL positive: 48 hr cold: 2+/-0.6%, 48 hr cold and 24 hr rewarming: 54+/-17%), which was confirmed by the TEM based on typical apoptotic features. Addition of 2-MAC and DFO significantly inhibited rewarming-induced apoptotic cell death (plus 2-MAC: 3+/-1%, plus DFO: 3+/-2%). CONCLUSION: Our study in human tubular cells provides evidence that cold storage per se does not result in apoptosis, but is primarily necrotic. However, rewarming is associated with significant apoptosis in the presence of ongoing necrosis, speculatively due to the activation of the apoptotic enzymic process of sublethally injured cells. Inclusion of antioxidants in the storage solution confers protection against both cold storage and rewarming-induced necrosis and apoptosis.