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D C Marsh

Publications and source records attributed to D C Marsh.

At least 19 recordsLinked to original sources
PubMed1996

Comparison of isolated hepatocytes and tissue slices for study of liver hypothermic preservation/reperfusion injury.

Simple models are needed that effectively test the variables that may be important in liver preservation. Two such models are isolated hepatocytes and tissue slices. In this study the effects of hypothermic preservation on the viability of hepatocytes (HC) and tissue slices (TS) from rat livers were measured by LDH leakage after cold storage and rewarming. We compared how glycine, calcium, and fasting, shown previously to affect preservation injury in hepatocytes, affected both HC and TS viability. Hepatocytes were cold-stored in University of Wisconsin organ preservation solution for up to 48 h and rewarmed in Krebs-Henseleit Bicarbonate (KHB) for 120 min. Tissue slices were studied in two ways. Either livers were cold-stored intact and then tissue slices (TS-A) prepared and rewarmed in KHB, or tissue slices were prepared from the fresh liver, cold-stored, and then rewarmed (TS-B). The latter method may be similar to cold storage of HC. Freshly prepared samples (HC, TS-A, or TS-B) showed < 15% LDH leakage during the rewarming phase. Cold storage for 24 h resulted in < 30% LDH leakage in all preparations. After 48 h cold storage there was a significant increase in LDH leakage (HC, 65.1 +/- 5.1%; TS-A, 52.9 +/- 0.8%, TS-B, 53.6 +/- 2.6%). Glycine (3 mM) or calcium (1.5 mM) included in the KHB significantly reduced LDH leakage from 48 h cold-stored HC to 20.7 +/- 1.8 and 26.3 +/- 2.4%, respectively. These agents caused a smaller decrease in LDH release from tissue slices (around 40%). Hepatocytes appear more susceptible to preservation/reperfusion damage than the more structurally intact tissue slices as suggested by the greater release of LDH. Another difference was that the agents which improved preservation quality of HC were not as effective in TS. Hepatocytes may be more vulnerable to preservation/reperfusion damage because of the harsh methods used in their preparation. The damage induced during preparation appears amenable to suppression by glycine or calcium. Tissue slices, which are intact pieces of liver tissue, may be more suitable for studies related to development of better methods for liver preservation. The intact cells in TS have not been exposed to harsh conditions and maintain a more natural cell-cell relationship.

Adenosine↗
PubMed1995

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

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

Adenosine↗
PubMed1994

Effect of glycine on isolated, perfused rabbit livers following 48-hour preservation in University of Wisconsin solution without glutathione.

Glycine has been shown to decrease membrane injury in isolated cells due to hypoxia or cold ischemia. The mechanisms of action of glycine are not known, but glycine may be useful in organ preservation solutions or in treating recipients of liver transplantation. In this study the isolated, perfused rabbit liver was used to measure how glycine affected liver performance after 48-h preservation in University of Wisconsin (UW) solution without added glutathione. UW solution is less effective for 48-h liver preservation when glutathione is omitted. Rabbit livers stored for 48 h without glutathione show a large increase in enzyme release (LDH and AST) from the liver and a reduction in bile production. The addition of 15 mM glycine to UW solution, in place of glutathione, did not improve bile production or reduce enzyme release. However, infusion of 10 mM glycine into the reperfused liver lowered LDH release significantly (from 2383 +/- 562 units/100 g to 1426 +/- 286 units/100 g) during the initial reperfusion of the 48-h preserved liver. Hepatamine, a parenteral nutrition solution containing glycine, as well as other amino acids, was also effective in lowering LDH release from the preserved liver. Although glycine reduced LDH release, it did not decrease the amount of AST released from the liver, nor did it improve bile production. Thus, we conclude that glycine, either in UW solution or given to the liver upon reperfusion, has no significantly beneficial effect as tested in this model. Further testing of glycine, however, should be conducted in an orthotopic transplant model in the rat or dog.

Adenosine↗
PubMed1993

Effect of fasting on hepatocytes cold stored in University of Wisconsin solution for 24 hours.

Although there have been improvements in liver preservation, liver dysfunction still remains a serious consequence of liver transplantation. This may be related to cold ischemic injury since the incidence of dysfunction increases with longer preservation times. However, even some livers preserved for short periods of time (less than 15 hr) develop liver dysfunction. One possible cause may be the lack of adequate nutritional support, and the donor may be exposed to prolonged periods of hyponutrition. In this study, we have compared the effects of fasting on functions of hepatocytes isolated from the rat. Hepatocytes were cold stored in University of Wisconsin solution for 24 hr and analyzed at the end of preservation as well as at the end of rewarming in Krebs-Henseleit buffer for 120 min. The glycogen content of fed cells was 1.57 mumol/mg protein and this was reduced by 95% in cells from fasted rats. After cold storage and rewarming, hepatocytes from fasted rats lost 84.2 +/- 2.5% of the total cellular lactate dehydrogenase versus only 32.7 +/- 3.8% (P < 0.001) in cells from fed rats. Also, ATP and reduced glutathione content of fasted cells were significantly reduced, free fatty acids were higher (P = 0.0154), and protein synthesis was reduced to 41% of controls (versus only 88% in fed cells), although there were no differences in phospholipid content. When hepatocytes from fasted rats were rewarmed in Krebs-Henseleit buffer containing fructose (10 mM), lactate dehydrogenase release was reduced from 80% to 34.4 +/- 0.2% and ATP content was significantly higher with fructose than without. Hepatocytes from fasted rats, therefore, are more sensitive to cold ischemic injury than cells from fed rats. The increased sensitivity appears related to the lack of glycogen as a source of substrates for metabolism during rewarming. This is supported by the fact that addition of fructose, which is metabolized readily by hepatocytes through glycolysis, suppressed rewarming injury to cells from fasted rats. The nutritional status of the donor, therefore, may play a pivotal role in the results of liver preservation and transplantation. Effective donor nutritional management may reduce the incidence of liver dysfunction after transplantation.

Adenosine↗
PubMed1993

Glycine protects hepatocytes from injury caused by anoxia, cold ischemia and mitochondrial inhibitors, but not injury caused by calcium ionophores or oxidative stress.

Isolated hepatocytes, suspended in an organ preservation solution, can be preserved at 4 degrees C for up to 6 days. After preservation, normothermic-normoxic incubation causes loss of hepatocyte viability. The addition of 3 mmol/L glycine to the rewarming medium prevents the loss of viability. In this study we investigated the cytoprotective effects of glycine under many conditions known to cause hepatocellular injury to understand the mechanism of cold-induced injury in the liver. Hepatocytes were suspended in modified Krebs-Henseleit buffer with or without 3 mmol/L glycine and exposed to agents or conditions known to induce cell death. Hepatocyte viability was assessed by measuring the percentage of lactate dehydrogenase leakage from the cells and the concentration of ATP during incubation at 37 degrees C under room air for up to 90 min. Mitochondrial inhibitors (potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone); calcium ionophores (ionomycin and A23187); an oxidizing agent, tert-butyl hydroperoxide; and anoxia were all used to cause cell injury. Hepatocytes were also isolated from fasted rats and hypothermically preserved as another model of cell death. Other amino acids were also tested in the hypothermic preservation model to study the specificity of the amino acid requirement for prevention of lactate dehydrogenase leakage. Of the amino acids tested, only alanine (10 mmol/L) and the combination of alanine (3 mmol/L) and serine (3 mmol/L) were as effective as glycine in preventing lactate dehydrogenase release in the hypothermic preservation model.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1992

Urea and protein synthesis in cold-preserved isolated rat hepatocytes.

We used an isolated-hepatocyte model to study how hypothermic storage (simulating liver preservation) affects metabolism after prolonged preservation. Rat hepatocytes were stored in the University of Wisconsin solution for up to 72 hr. After each day of storage, protein synthesis, urea synthesis, ATP content and lactate dehydrogenase release were determined in rewarmed (37 degrees C) and oxygenated hepatocytes. Protein synthesis ([3H]-leucine incorporation into protein) was depressed by 16% +/- 4%, 54% +/- 6% and 69% +/- 4% after 24 hr, 48 hr and 72 hr, respectively. Urea synthesis, ATP synthesis and lactate dehydrogenase release were similar to those in control hepatocytes (no preservation). Fasting of the rats before isolation of hepatocytes caused more rapid loss of protein-synthesis capabilities (59% in 24 hr) with no significant loss of lactate dehydrogenase, urea synthesis or ATP synthesis. Hepatocyte viability (lactate dehydrogenase release) as judged by membrane permeability, ATP synthesis and potassium content can be maintained after up to 6 days of cold storage. However, protein synthesis is depressed after only 48 hr of cold storage. Thus hypothermic storage of the liver causes a change in the metabolic capabilities of the hepatocytes, and the timing of the loss of protein synthesis is similar to the limits of successful cold storage of the whole liver (48 hr). Thus a limit to long-term storage of the liver may be related to loss of protein synthesis. In liver transplantation, one indication of poor preservation is a decrease in serum albumin and clotting factors with increased tissue edema and bleeding diathesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1991

The role of oxygen free radicals in organ preservation.

There is controversy over the role of oxygen free radical-induced damage in preserved organs following reperfusion. Furthermore, there has been no definitive study that shows a dramatic improvement in organ functions, delayed graft functions, or improved longevity in organ transplants with oxygen free radical scavenger therapy. However, the presence of glutathione in a new organ preservation solution (University of Wisconsin, UW, solution) yields improved preservation of the liver and heart. The beneficial effect of glutathione may involve in scavenging of cytotoxic products of oxygen metabolism. The results discussed here show that glutathione improves liver preservation. Also, it is shown that glycine, and amino acid component of glutathione, can also give cytoprotection to the rabbit and dog liver tested by either isolated perfusion or orthotopic transplantation. Thus, there may be an involvement of oxygen free radicals in damage to organs hypothermically preserved and transplanted. The injury may occur within the cells or may be due to oxygen within the cells or may be due to oxygen free radicals generated in the extracellular environment.

Adenosine↗
PubMed1991

Glycine prevention of cold ischemic injury in isolated hepatocytes.

Isolated hepatocytes suspended in a liver preservation solution (University of Wisconsin (UW) solution) and exposed to cold (5 degrees C) ischemia lose viability (LDH release) after 3 (76.5 +/- 2.6% extracellular LDH) and 4 days (90.3 +/- 5.7% extracellular LDH) storage when rewarmed (37 degrees C) in Krebs-Henseleit buffer. However, if 3 mM glycine is added to Krebs-Henseleit buffer the loss of LDH on rewarming was suppressed (% LDH = 24.4 +/- 2.2% and 33.2 +/- 3.0%, at 3 and 4 days, respectively). The protection by glycine could also be obtained by storing the hepatocytes in the UW solution containing 15 mM glycine and rewarming in the absence of glycine in Krebs-Henseleit buffer. There did not appear to be a relationship between the protection by glycine and glutathione concentration of the hepatocytes as shown by the lack of effect of a glutathione synthetase inhibitor (butathionine sulfoximine) on the protective effects of glycine. Other amino acids did not provide protection to hepatocytes exposed to cold ischemia. The mechanism of action of glycine is not known, but this compound may be important in improving cold storage of livers for transplantation.

Animals↗
PubMed1991

Hypothermic preservation of hepatocytes. III. Effects of resuspension media on viability after up to 7 days of storage.

Hepatocyte suspensions provide a rapid method to determine how hypothermic storage affects liver cell metabolism and viability. Using these studies, improved methods of hypothermic liver preservation for transplantation may be developed. In this study, rat hepatocytes were cold-stored for up to 7 days in University of Wisconsin liver preservation solution. At the end of each day of storage hepatocytes were resuspended in Krebs-Henseleit buffer or tissue-culture medium (Liebovitz-15; Fischer's; modified Fischer's, which was similar to Fischer's but with glycine and cysteine added; or Waymouth's medium). Hepatocyte viability was assessed by rewarming and oxygenating the suspensions and measuring the percentage of leakage of lactate dehydrogenase from the cells, the cellular concentration of potassium and the stimulation of respiration by succinate, all measures of plasma membrane integrity. Additionally, concentrations of ATP and glutathione after rewarming and reoxygenation in the various resuspension media were measured. Hepatocyte permeability to lactate dehydrogenase did not increase during cold storage of 1 to 7 days (7.2% +/- 2% leakage), indicating that most of the hepatocytes remained viable during cold storage. However, when rewarmed, loss of viability (leakage of lactate dehydrogenase) was dependent on the composition of the resuspension media. In Krebs-Henseleit buffer, viability was reduced after 2 and 3 days of storage (lactate dehydrogenase leakage on rewarming = 70% to 90%). Leakage of lactate dehydrogenase was reduced significantly after resuspension in tissue-culture media. After 6 days of storage, lactate dehydrogenase leakage from hepatocytes stored in Liebovitz- 15 or modified Fischer's was only about 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Hypothermic preservation of hepatocytes. II. Importance of Ca2 and amino acids.

The importance of the components of a tissue culture media, Leibovitz-15 (L-15), for maintaining viability of hypothermically preserved hepatocytes was analyzed. Hepatocytes isolated from rat livers were incubated at 5 degrees C in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). L-15 + 5 g% polyethylene glycol (PEG) or variants of this solution were used as the preservation media. After 48 hr of storage, hepatocyte viability was assessed by measuring the release of LDH into the incubation medium and cell volumes were determined. Following 90 min of normothermic incubation (to simulate organ reperfusion), mitochondrial function was measured. Hepatocytes stored in the complete L-15 solution were about 90% viable at the end of 48 hr of storage, while cells stored in a solution containing only the principle electrolytes (PE) lost viability (70% viable). Only the addition of a combination of divalent cations (Ca/Mg) and amino acids was sufficient to maintain viability equivalent to that obtained in the complete L-15 mixture. Hepatocytes suspended in L-15 maintained normal cell volumes (3.85 microliters/mg protein), while cells in the PE solution were swollen with cell volumes of 4.66 microliters/mg protein. Only the addition of Ca/Mg to the PE solution was effective at suppressing cell swelling similar to the complete L-15 media. Both basal and uncoupler-stimulated respiration were depressed in cells stored in the PE solution (15 and 28 nmol O2/min/mg protein) as compared to cells in L-15 (21 and 41 nmol O2/min/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1989

The effect of pH on the viability of hypothermically stored rat hepatocytes.

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).

Animals↗
PubMed1989

Hypothermic preservation of hepatocytes. I. Role of cell swelling.

Hepatocytes from isolated rat livers were hypothermically incubated (5 degrees 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 hypothermic 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.

Animals↗
PubMed1987

Cholinoceptor-mediated mechanical and electrical responses of rat oesophageal striated musculature. A comparison of two in vitro methods.

1. Both muscarinic and nicotinic cholinoceptor agonists produce a contractile response of the rat oesophageal tunica muscularis externa (TME) in vitro. 2. The muscarinic receptor-mediated responses were confined to the distal 0.5 cm of the TME, which contains an admixture of smooth muscle. 3. Nicotinic receptor-mediated responses were obtained throughout the length of the TME striated musculature and consisted of a fast and slow component, representing TTX-sensitive contractions, associated with muscle action potentials, and electrically silent contractures, respectively. 4. Contractions were observed only in superfused preparations. 5. Contractures were resistant to changes in extracellular cation concentrations designed to eliminate generation of nerve and muscle action potentials or release of neurotransmitters.

Animals↗