Preservation of pig liver allografts after warm ischemia: normothermic perfusion versus cold storage

Warm ischemia is known to induce substantial damage to the liver parenchyma. With respect to clinical liver transplantation, the tolerance of the liver to warm ischemia and the preservation of these organs have not been studied in detail. In isolated reperfused pig livers we proceeded according to the following concept: Livers were subjected to 1 or 3 h of warm ischemia. Subsequently, these organs were preserved by either normothermic perfusion or cold storage (histidine-tryptophan-alpha-ketoglutarate, HTK) for 3 h each. After storage, liver function was assessed in a reperfusion circuit for another 3 h. Parameters under evaluation were bile flow, perfusion flow, oxygen consumption, enzyme release into the perfusate (creatine kinase, glutamic oxaloacetic transaminase (GOT), lactic dehydrogenase, and glutamic pyruvic transaminase), and histomorphology. Damage to the liver was lowest after warm ischemia of 1 h. The results after cold storage were superior to those after normothermic perfusion (GOT: 3.2 +/- 0.3 and 2.6 +/- 0.2 U/g liver; cumulative bile production: 14.7 +/- 2.1 and 9.4 +/- 1 ml, respectively; P < 0.05). In contrast, we found substantial damage at the end of reperfusion in livers undergoing 3 h of warm ischemia under both preservation techniques with severe hepatocellular pyknoses and essentially altered nonparenchymal cells. The results suggest that pig livers undergoing 1 h of warm ischemia and cold storage for 3 h with HTK solution may lead to functioning after transplantation.     

Background and prognostic implications of perireperfusion tissue injuries in human liver transplants: a panel histochemical study

BACKGROUND: Hepatic graft reperfusion is associated with inflammatory processes of unknown relevance to the fate of graft. This study aimed to clarify this relevance by histochemical analyses of human hepatic grafts. METHODS: Paired tissue samples were taken at the end of cold preservation and 2 hr after reperfusion (n=39). From six additional grafts, biopsies were performed at the end of cold preservation only. Injury or inflammatory markers of sinusoidal endothelium (von Willebrand factor-related antigen [vWF]), Kupffer cells (25F9), platelets (CD62), neutrophil leukocytes (CD11b), interleukin (IL)-1beta, intercellular adhesion molecule (ICAM)-1, and HLA-DR were evaluated semiquantitatively by indirect immunoperoxidase staining. Steatosis was also evaluated by hematoxylin and eosin staining. RESULTS: vWF, CD62+ platelet aggregation, CD11b+ leukocytes, and IL-1beta levels increased after reperfusion, and these levels correlated with prereperfusion levels. Not only vWF, CD62+ platelets, CD11b+ leukocytes, IL-1beta, ICAM-1, and steatosis after reperfusion, but also IL-1beta, ICAM-1, and steatosis before reperfusion correlated with postoperative peak transaminase. Furthermore, vWF, CD11b+ leukocytes, 25F9+ macrophages, and ICAM-1 after reperfusion were associated with primary graft nonfunction and strong expressions of ICAM-1 or HLA-DR with early acute rejection. Although some markers (IL-1beta, CD62+ platelets, and CD11b+ leukocytes) correlated with preharvesting parameters (donor age or length of intensive care unit stay), none showed any significant correlation with cold preservation. CONCLUSION: Synergistic inflammatory events in the hepatic graft at reperfusion, which have a significant impact on the later clinical course, are largely defined and precipitated by injury or activation of nonparenchymal cells preceding reperfusion or even graft harvesting.     

Enhancement of endogenous cyclic AMP signal: a new approach to allow for cold preservation of rat livers from non-heart-beating donors?

BACKGROUND: The organ donor shortage has led to a reconsideration of the use of non-heart-beating donors (NHBDs). However, graft injury due to warm ischemia in NHBD livers strongly affects posttransplant outcome. The present study was aimed at investigating the role of the cellular cyclic (c)AMP second messenger signal with regard to hepatic viability after cold preservation of NHBD livers. METHODS: Cardiac arrest was induced in Wistar rats by frenotomy of the anesthetized nonheparinized animal. After 30 min, the livers were excised and flushed with 20 ml of heparinized saline solution, rinsed with 10 ml of University of Wisconsin (UW) solution, and stored submerged in UW solution at 4 degrees C for 24 hr. In half of the experiments, UW solution was supplemented with glucagon (0.5 microg/ml) to increase the cAMP signal in the liver. Reperfusion was carried out in vitro after all livers were incubated at 25 degrees C in saline solution to replicate the period of slow rewarming during surgical implantation in vivo. RESULTS: Hepatic levels of cAMP (nmol/g dry weight) declined from 1.21+/-0.05 to 0.53+/-0.03 (P<0.01) at 30 min after cardiac arrest. Subsequent storage in UW solution resulted in a further decline to 0.35+/-0.04 after 24 hr in group A, whereas glucagon treatment enhanced cellular cAMP signal to 0.64+/-0.06 (P<0.01). Upon reperfusion, liver integrity was significantly improved after glucagon administration, with 66% reduction in alanine aminotransferase release and a threefold increase in hepatic bile production as compared with untreated livers. Moreover, liver ATP tissue levels were restored to only 2.19+/-0.51 micromol/g in the untreated group but reached 4.97+/-0.41 micromol/g (P<0.05) after treatment with glucagon. CONCLUSIONS: Posthoc conditioning of predamaged livers by glucagon enhances cAMP tissue levels during ischemic preservation and improves hepatic integrity upon reperfusion. This may represent a promising approach for the use of livers from non-heart-beating donors in clinical transplantation.     

The effects of thyroid hormone modulation on rat liver injury associated with ischemia-reperfusion and cold storage

We investigated the effects of thyroid hormone modulation on liver injury associated with ischemia-reperfusion (I-R) and cold storage in rats. First, euthyroid and thyroxine (T4)-pretreated rats were exposed in vivo to 20-min global liver ischemia, then 30-min reperfusion. Liver injury was assessed by measuring serum alanine aminotransferase (ALT) levels. Liver concentrations of adenine nucleotides, reduced glutathione (GSH), and oxidized glutathione were evaluated. Second, rats were given the antithyroid drug propylthiouracil (PTU). Livers stored at 0-1 degrees C in Euro-Collins' solution for 20 h were reperfused at 37 degrees C for 15 min. Lactate dehydrogenase (LDH) in the effluent perfusate and bile flow were evaluated during reperfusion. Serum ALT levels increased after ischemia and I-R. ALT increased significantly more in T4-pretreated than in euthyroid rats after ischemia and I-R. Preischemic levels of adenosine triphosphate (ATP) were significantly lower in livers from T4-pretreated than in euthyroid rats (6.22 +/- 0.7 and 11 +/- 0.9 nmol/mg protein, respectively; P < 0.05). After ischemia, liver ATP was similarly reduced in T4-pretreated and euthyroid rats. After reperfusion, ATP partially recovered in euthyroid rats but remained low in T4-pretreated rats (6.7 +/- 1.0 and 1.91 +/- 0.7 nmol/mg protein, respectively; P < 0.05). Preischemic levels of liver GSH decreased to 44% in T4-pretreated rats. After ischemia, GSH decreased similarly in euthyroid and T4-pretreated rats. GSH recovered promptly after reperfusion in euthyroid rats but remained low in T4-pretreated rats (13.9 +/- 3.3 and 3.9 +/- 0.9 nmol/mg protein, respectively; P < 0.02). During reperfusion after cold storage, LDH in effluent perfusate was significantly lower and bile flow higher in livers from PTU-pretreated rats than from euthyroid rats. The histopathological changes observed after I-R and cold storage confirmed the biochemical findings. Our results suggest that T4 administration exacerbates pretransplant liver damage by increasing liver susceptibility to I-R, whereas PTU administration reduces the liver injury associated with cold storage. Implications: We studied the effects of thyroid hormone modulation on liver injury associated with ischemia-reperfusion and cold storage in rats. Thyroxine administration increased susceptibility to ischemia-reperfusion injury, whereas the antithyroid agent propylthiouracil reduced the deleterious effects associated with cold storage.     

Gentle in situ liver manipulation during organ harvest decreases survival after rat liver transplantation: role of Kupffer cells

BACKGROUND: The etiology of primary graft nonfunction and dysfunction is unknown but most likely involves Kupffer cell-dependent reperfusion injury. However, the donor operation and surgical technique may also have an effect on the outcome after transplantation. Because liver manipulation during harvest cannot be prevented completely with standard procedures, its effect on survival was assessed here. METHODS: Donor livers were harvested from female Sprague-Dawley rats (200-230 g). Briefly, after minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 subsequent minutes. At 25 min, all livers were perfused with cold University of Wisconsin solution via the portal vein, and transplantation was performed after cold storage (1 hr). In some rats, Kupffer cells were destroyed with gadolinium chloride or inactivated with dietary glycine before harvest. Survival, proteolytic activity in the rinse effluent, serum transaminases, trypan blue distribution to index microcirculation, and histology were compared. RESULTS: In the nonmanipulated group, survival was 100% after transplantation; however, gentle manipulation decreased survival by 70%. Further, manipulation elevated transaminases fivefold and caused about 200% necrosis. At harvest, proteolytic activity and the time for trypan blue to distribute homogeneously were elevated three- to eightfold by manipulation. Gadolinium chloride and glycine prevented the effects of manipulation on all parameters studied. CONCLUSION: These data indicate for the first time that brief, gentle manipulation of the donor liver has a marked detrimental effect on survival by priming or activating Kupffer cells. This may represent an important early event in pathogenesis, because Kupffer cells play an important role in primary graft nonfunction.     

Phospholipid signaling in leukocytes

The principal cause of primary non-function in orthotopic liver transplantation is thought to be preservation injury to the microvasculature. We, therefore, evaluated if effluent levels of hyaluronate, whose uptake is an endothelial cell marker, could predict early graft function and ultimate graft outcome in orthotopic liver transplantation. A total of 102 cases were studied in two phases. In the first phase, we attempted to determine if a correlation existed between effluent hyaluronate levels, early graft function and ultimate graft outcome. This phase of the study was also used to determine hypothetical cut-off values for hyaluronate which could discriminate between good and bad livers. Thirty-two livers orthotopically transplanted to randomly selected primary recipients were studied. After varying periods of static cold storage (4 degrees C) in University of Wisconsin solution, the livers were reinfused with cold (4 degrees C) lactated Ringer's solution. The first 50 ml of the reperfusion effluent was collected from the infrahepatic vena cava. Effluent samples were analyzed for hyaluronate. Linear regression analysis demonstrated a significant correlation between effluent hyaluronate levels and post-operative aspartate and alanine aminotransferase levels (p < 0.001 for both). Logistic regression demonstrated a highly significant correlation (p = 0.0056) between effluent hyaluronate levels and ultimate graft outcome. Generation of Receiver Characteristics Curves indicated that a level between 400 and 430 micrograms.l-1 could possibly discriminate between good livers and those at risk of early graft failure. The authenticity of this hyaluronate cut-off level was further confirmed in the second phase of the study where 70 consecutive primary crossmatch-negative transplants were performed. A highly significant difference was observed in peak aspartate and alanine aminotransferase levels in the first week (p < 0.0006 and p < 0.0005, respectively) between livers with effluent hyaluronate levels < or = 400 micrograms.l-1 and livers with hyaluronate levels higher than 400 micrograms.l-1. Logistic regression revealed a highly significant correlation between effluent hyaluronate levels and graft success (p = 0.0001). Since hyaluronate uptake by the microvascular endothelial cell is significantly greater than production, high hyaluronate effluent levels in failed livers would be due to decreased hyaluronate uptake by the injured microvascular endothelial cell. We therefore conclude that effluent hyaluronate levels may prove to be a reliable preoperative test to assess early graft function and outcome in clinical orthotopic liver transplantation.     

Interaction of nitric oxide and endothelin-1 in ischemia/reperfusion injury of rat heart

Although several studies have demonstrated that nitric oxide appears to be cardioprotective and endothelin-1 (ET-1) deleterious in myocardial ischemia/reperfusion injury, their interactions in the intact heart are unknown. Therefore, coronary effluent and interstitial fluid ("transudate") levels of ET-1 and cyclic GMP, an indirect measure of nitric oxide production, were determined simultaneously in normoxic and reperfused hearts and compared with myocardial and coronary function. Rat hearts were buffer-perfused at 9 ml/min/g heart wet weight for 45 min (baseline), followed either by another 45 min of perfusion (normoxia), or 15 min of total global ischemia and 30 min reperfusion. Hearts received, from 42-90 min, either vehicle, the inhibitor of nitric oxide formation NG-nitro-L-arginine (L-NNA; 200 micromol/l), the nitric oxide donor S-nitroso-N-acetyl-DL-penicillamine (SNAP; 200 micromol/l), or the ET receptor antagonist PD 142893 (200 nmol/l). Both mediators were released preferentially into the vascular lumen which resulted in similar luminal and interstitial concentrations of cyclic GMP, but three-fold higher levels of ET-1 in tissue because of the higher effluent than transudate flow rate. L-NNA increased the release of ET-1 and worsened coronary function, whereas SNAP had opposite effects. On reperfusion, considerable functional impairment was observed, although levels of cyclic GMP both in the vascular and tissue compartment were not reduced, but even increased. Reperfusion functional impairment was aggravated after inhibiting the synthesis of nitric oxide, whereas SNAP restored cardiac and coronary function close to pre-ischemic level. Deterioration of function corresponded with an increased level, and improvement with a decreased level of intersitial ET-1 at the onset of reperfusion. PD 142893 was similarly cardioprotective as SNAP both in normoxia and reperfusion. These results suggest that in reperfusion, cardiac function is depressed, despite increased rather than decreased endogenous nitric oxide production, largely due to the prevalence of the deleterious effects of ET-1 which are overcome by antagonism of ET receptors or exogenous nitric oxide supplied by SNAP.     

Hypothermic ischaemia of the liver: a re-perfusion phenomenon

BACKGROUND: The effects of hypothermic injury to the liver were investigated on an isolated perfusion circuit by comparing porcine livers with varying degrees of preservation injury. METHODS: A group of unstored livers (n = 5) were compared to livers stored in University of Wisconsin (UW) solution for 18 h (n = 5), and a group of livers stored in Hartmann's solution for 18 h (n = 5). RESULTS: We observed that the degree of platelet sequestration was directly related to the severity of the preservation injury. After 2 h of isolated liver perfusion, the perfusate platelet count fell from 148 +/- 14 x 10(9)/L to 84 +/- 13 x 10(9)/L for control livers. In comparison for livers stored in UW solution, the platelet count fell from 173 +/- 43 x 10(9)/L to 61 +/- 14 x 10(9)/L representing a 64.8% fall, while for those stored in Hartmann's solution, an even more profound fall from 152 +/- 36 x 10(9)/L to 19 +/- 9 x 10(9)/L (87.5% fall) was observed. The difference between the UW-stored and Hartmann's-stored livers was significant (P < 0.05). However, using this model, the degree of leukocyte sequestration did not differentiate the groups. Both histological and ultrastructural examination of liver biopsies taken immediately following revascularization demonstrated that for mild degrees of preservation injury following hypothermic storage, changes occur to the sinusoidal lining cells well before changes to the parenchymal elements. CONCLUSIONS: These findings substantiate the hypothesis that the primary injury associated with hypothermia involves the sinusoidal lining cells (non-parenchymal elements), that it is predominantly a reperfusion phenomenon and that efforts at improving preservation should therefore be targeted primarily at these cells and not the hepatocytes.     

Anion channels and the stimulation of anthocyanin accumulation by blue light in Arabidopsis seedlings

The effects of endothelin 1 (ET-1) on hemodynamics and acute liver damage were studied using perfused livers of rats treated with D-galactosamine. In control liver perfused in situ with constant pressure, infusion of ET-1 into the portal vein at a concentration of 0.1 nmol/L decreased the flow rate without a significant leakage of lactate dehydrogenase (LDH) or aspartate transaminase (AST) into the effluent. In contrast, in similarly perfused liver 24 hours after treatment with D-galactosamine (800 mg/kg intraperitoneally), ET-1 caused rapid and remarkable increases in the leakage of LDH and AST from the liver accompanied by the reduction of perfusion flow to the extent similar to that observed in control livers. In addition, ET-1 decreased oxygen uptake and bile secretion in galactosamine-treated livers. The potentiating effects of ET-1 on enzyme leakage were also observed under constant flow conditions. Moreover, infusion of the thromboxane A2 analogue at a concentration of 10 nmol/L decreased the flow rate markedly, yet the rapid increases in enzyme leakage were not observed. Infusion of ET-3 induced the responses of flow reduction and the potentiation of rapid enzyme leakage similar to those obtained with ET-1. Neither the endothelin A-receptor antagonist BQ485 nor the endothelin B-receptor antagonist BQ788 could inhibit the acute liver damage caused by ET-1; instead they exaggerated its effects. The combination of both antagonists together, however, almost completely suppressed the flow reduction and the potentiation of enzyme leakage caused by ET-1. These results indicate that ET-1 is capable of aggravating acute liver damage not merely through reduction of the flow rate but through direct action on liver cells. They also suggest that both the endothelin A and endothelin B receptors are involved in this action of ET-1.     

IL-6 produced by Kupffer cells induces STAT protein activation in hepatocytes early during the course of systemic listerial infections

Kupffer cells were the principal source of IL-6 produced in the livers of mice following i.v. inoculation of Listeria monocytogenes. IL-6 mRNA expression and the production of IL-6 were reduced drastically within the nonparenchymal liver cell population derived from mice rendered Kupffer cell depleted by pretreatment with liposome-encapsulated dichloromethylene diphosphonate. A sharp increase in the appearance of activated STAT3 occurred in extracts of purified hepatocytes derived from normal mice infected i.v. with Listeria. Remarkably, the kinetics of this increase overlapped IL-6 mRNA expression by Kupffer cells; each peaked at approximately 30 min postinfection. No increase in STAT3 activation was observed in IL-6-deficient or Kupffer cell-depleted animals. The results of these experiments indicate that the synthesis of IL-6 and the activation of STAT3 within hepatocytes are critical functions of Kupffer cells occurring very early during the course of systemic listerial infections.     

Endothelin-1 increases susceptibility of isolated rat hearts to ischemia/reperfusion injury by reducing coronary flow

Endothelin-1 (ET-1) is the most potent vasoconstrictor known to date, and it was proposed that this peptide plays a major role in myocardial ischemia/reperfusion injury. ET-1 could increase myocardial susceptibility to ischemia by two mechanisms: via coronary flow reduction and/or via direct, metabolic effects on the heart. In isolated, buffer-perfused rat hearts, function was measured with a left ventricular balloon, and energy metabolism (ATP, phosphocreatine, inorganic phosphate, intracellular pH) was estimated by 31NMR-spectroscopy. Under constant pressure perfusion, hearts were subjected to 15 min of control perfusion, 15 ("moderate injury") or 30 ("severe injury") min of global ischemia, followed by 30 min of reperfusion. Hearts were pre-treated with ET-1 (boluses of 0.04, 4, 40 of 400 pmol) 5 min prior to ischemia. In the control period, ET-1 reduced coronary flow, ventricular function, phosphocreatine and intracellular pH dose-dependently: during ischemia/reperfusion, coronary flow, functional recovery and high-energy phosphate metabolism were adversely affected by ET-1 in a dose-related manner. To study effects of ET-1 not related to coronary flow reduction, additional hearts were perfused under constant flow conditions (ET-1 0 or 400 pmol) during 15 min of control, 15 min of ischemia and 30 min of reperfusion. When coronary flow was held constant, functional and energetic parameters were similar for untreated and ET-1 treated hearts during the entire protocol, i.e. the adverse effects of ET-1 on function and energy metabolism during ischemia/reperfusion were completely abolished. In both constant pressure and constant flow protocols, 400 pmol ET-1 reduced the extent of ischemic intracellular acidosis. The authors conclude that ET-1 increases the susceptibility of isolated hearts to ischemia/reperfusion injury via reduction of coronary flow.     

Role of glutathione in hepatic bile formation during reperfusion after cold ischemia of the rat liver

BACKGROUND/AIMS: Liver reperfusion following cold ischemia is frequently associated with diminished bile flow in patients undergoing liver transplantation. Glutathione is a major determinant of bile-acid independent bile flow, and the effects of cold ischemia on biliary glutathione excretion are unknown. METHODS: We examined the effects of cold ischemia (University of Wisconsin solution (4 degrees C), 24 h) with subsequent reperfusion (100 min) on biliary glutathione excretion in a recirculating system. Since glutathione might represent an important antioxidant within the biliary tract and oxidative stress in the biliary tract during reperfusion could contribute to the pathogenesis of bile duct injury after liver transplantation, we also assessed bile duct morphology in reperfused livers of mutant TR- -rats, in whom biliary excretion of glutathione is already impaired. RESULTS: Hepatic bile formation was diminished in reperfused Wistar rat livers after cold ischemia. Biliary glutathione concentrations and output were significantly decreased and correlated with postischemic changes in bile secretion. An increased biliary oxidized glutathione/glutathione ratio, indicating oxidative stress, was detected only immediately after the onset of reperfusion. Basal bile flow rates in TR- -rat livers which were already markedly reduced in control-perfused livers, decreased further during the early but not the later reperfusion period. Reperfusion of both Wistar and TR- -rat livers was not associated with electron microscopic evidence of bile duct damage. CONCLUSIONS: We conclude that impaired biliary excretion of glutathione contributes to decreased bile flow after cold ischemia. The absence of biliary glutathione does not appear to promote ultrastructural evidence of bile duct injury during reperfusion in the isolated perfused rat liver.     

Antiangiogenic agents protect liver sinusoidal lining cells from cold preservation injury in rat liver transplantation

BACKGROUND & AIMS: Low temperature preservation causes unique liver injuries to the sinusoidal lining cells characterized by endothelial cell detachment and rounding and Kupffer cell activation. These changes are similar to those observed during the early stages of angiogenesis. The aim of this study was to investigate if cold preservation injury is caused by the activation of angiogenic mechanisms. METHODS: Livers were obtained from rats pretreated with three well-known antiangiogenic agents (minocycline, interferon alfa-2b, and fumagillin) and were stored for various durations in cold preservation solutions. The effects of the drugs were evaluated by morphometric assessment of endothelial cell injury in H&E, trypan blue, and immunostained (TIE2/Tek) biopsy specimens. Graft functions and survival were evaluated in isolated perfused rat liver and arterialized orthotopic liver transplantation models. RESULTS: Sinusoidal lining cell integrity and viability were significantly improved in animals pretreated with the drugs. Reperfusion injury and survival were also better in pretreated animals. Interferon alfa was the most potent agent, reducing injury even in livers preserved in the current most commonly used solution (University of Wisconsin solution). CONCLUSIONS: Cold preservation injury of liver may be the results of angiogenic mechanisms. This novel observation provides a rationale for improved liver preservation using antiangiogenic agents.     

Systemic liberation of interleukin-1 beta and interleukin-1 receptor antagonist in the perioperative phase of liver transplantation

We measured systemic serum levels of interleukin-1 receptor antagonist (IL-1ra), interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6) during the preoperative, anhepatic, and postreperfusional phases up to the 7th postoperative day in 60 patients undergoing orthotopic liver transplantation (LTx). In contrast to IL-1 beta, IL-1ra, TNF-alpha, and IL-6 showed a significant elevation in relation to the early phase after reperfusion, while TNF-alpha displayed a high grade of scatter. In addition, IL-1ra levels were significantly elevated during the anhepatic phase. Maximum serum levels were found at 15 min after reperfusion, 120 min after reperfusion, and on the 1st postoperative day, respectively. Serum levels decreased considerably at 24 h and 7 days after reperfusion. The comparative monitoring of systemic cytokine and cytokine antagonist levels, in particular the liberation of IL-1ra and IL-6 may provide useful parameters for the development of new liver preservation theories for LTx.     

Effect of temperature in long-term preservation of vascular endothelial and smooth muscle function

BACKGROUND: In clinical transplantation the donor organ is perfused with a cold preservation solution to obtain quick core cooling and a suitable environment for the tissue cells. Without good preservation of the vasculature, progressive deterioration of the blood flow during reperfusion may ultimately lead to the no-reflow phenomenon, even though the function of the other cells in the organ may be adequately preserved. The aim of this study was to find the optimal storage temperature for preservation of the vasculature. METHODS: The infrarenal aorta of 126 Sprague-Dawley rats were studied in organ baths: as fresh controls, after 36 hours of storage at 0.5 degrees C, 4 degrees C, 8.5 degrees C, and 22 degrees C in University of Wisconsin solution, and after 36-hour storage followed by transplantation and a lapse of 2 hours, 24 hours, and 7 days. The thromboxane analogue U-46619 was used to test contractility. Acetylcholine was used to elicit endothelium-dependent relaxation (EDR), and papaverine to elicit endothelium-independent relaxation. RESULTS: Storing the vessels at 0.5 degree C proved best regarding preservation of contractility, with a nonsignificant decrease, whereas storage at 4 degrees C and 8.5 degrees C resulted in a significant decrease after 36 hours. The contractility did not recover within 24 hours of in vivo reperfusion, but full recovery was seen after 7 days. Regardless of the preservation temperature used, a significant impairment in EDR was seen after 36 hours of storage. Two hours after transplantation, vessels stored at 4 degrees C and 8.5 degrees C showed no significant impairment in EDR, whereas those stored at 0.5 degrees C demonstrated a significant loss of EDR. After 24 hours and after 7 days, EDR was normal in all groups. CONCLUSIONS: Endothelium-dependent relaxing factor function is best preserved at 4 degrees C and 8.5 degrees C, whereas preservation of vascular smooth muscle function is best preserved at 0.5 degrees C.     

A fish oil diet minimizes hepatic reperfusion injury in the low-flow, reflow liver perfusion model

In this study, the effects of fish oil treatment on hepatic reperfusion injury in a low-flow, reflow perfusion model were investigated. Rats were fed powdered diets containing either 5% corn or 5% encapsulated fish oil for 13 to 15 days. Average daily food intake in both control and fish oil groups was about 20 g per rat, and weight gain averaged 9 g/rat/d. Livers were perfused at flow rates around 1 muL/g/min for 75 minutes, which caused cells in pericentral regions to become anoxic because of insufficient delivery of oxygen. When normal flow rates (about 4 mL/g/min) were restored for 40 minutes, reperfusion injury occurred. Upon reflow, lactate dehydrogenase (LDH) release increased from basal levels around 1 to 50 IU/g/h in livers from control rats, whereas fish oil treatment minimized values to 16 IU/g/h Rates of bile production reached 23 muL/g/h during reperfusion in livers from controls and 38 muL/g/h in the fish oil-treated group. Oxygen uptake was about 110 mumol/g/h during the reperfusion period in livers from both groups. Malondialdehyde (MDA), an end product of lipid peroxidation, was released into the effluent perfusate at rates around 80 nmol/g/h during reperfusion in controls, and values were not affected by fish oil treatment, Portal pressure, an indicator of hepatic microcirculation, increased from basal levels of 3 to 10.5 cm H2O during reperfusion in controls, but was only elevated to 8.3 cm H2O in the fish oil-treated group. In addition, Trypan Blue distribution time, another indicator of hepatic microcirculation, was reduced significantly by 44% by fish oil treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation of lung graft reperfusion injury by a nitric oxide donor

OBJECTIVE: One of the primary features of ischemia-reperfusion injury is reduced production of protective autocoids, such as nitric oxide, by dysfunctional endothelium. Administration of a nitric oxide donor during reperfusion of lung grafts may therefore be beneficial through modulation of vascular tone and leukocyte and platelet function. METHODS: Rat lung grafts were flushed with University of Wisconsin solution and reperfused for 1 hour in an ex vivo model incorporating a support animal. Group I grafts (n = 6) were reperfused immediately after explantation, group II (n = 6) and III (n = 5) grafts after 24 hours of storage at 4 degrees C. In group III, glyceryl trinitrate, a nitric oxide donor, was administered during the first 10 minutes of reperfusion at a rate of 200 micrograms/min. In an additional group (n = 5), 200 micrograms/min hydralazine was administered instead, to assess the effect of vasodilation alone. RESULTS: Graft function in group II deteriorated compared with that in group I, with significant reduction of graft effluent oxygen tension and blood flow and elevation of pulmonary artery pressure, peak airway pressure, and wet/dry weight ratio. In contrast, in group III, glyceryl trinitrate treatment improved graft function to baseline levels in all these parameters. Administration of hydralazine, meanwhile, produced mixed results with only two out of five grafts functioning at control levels. CONCLUSIONS: In this model, administration of glyceryl trinitrate to supplement the nitric oxide pathway in the early phase of reperfusion has a sustained beneficial effect on lung graft function after 24-hour hypothermic storage, probably through mechanisms beyond vasodilation alone.     

A comparison of the new preservation solution Celsior to Euro-Collins and University of Wisconsin solutions in lung reperfusion injury

BACKGROUND: The lung is particularly susceptible to reperfusion injury, both experimentally and clinically after transplantation. The extracellular-type preservation solution Celsior, which has been predominantly studied in cardiac preservation, has components designed to prevent cell swelling, free radical injury, energy depletion, and calcium overload. Using an isolated blood-perfused rat lung model, we investigated whether Celsior would decrease preservation injury and improve lung function after cold ischemic storage and reperfusion compared to Euro-Collins (EC) and University of Wisconsin (UW) solutions. METHODS: Lewis rat lungs were isolated, flushed with the respective cold preservation solution, and then stored at 4 degrees C for 6 or 12 hr. After ischemic storage, the lung block was suspended from a force transducer, ventilated with 100% O2, and reperfused for 90 min with fresh blood via a cannula in the pulmonary artery. Lung compliance, alveolar-arterial oxygen difference, and outflow oxygen tension were all measured. The capillary filtration coefficient (Kf), a sensitive measure of changes in microvascular permeability, was determined. RESULTS: For 6 hr of cold storage, lungs stored in Celsior had lower Kf values than those stored in EC, indicating decreased microvascular permeability. No other significant differences were noted between Celsior and EC or UW. For 12 hr of cold storage, Celsior provided increased oxygenation, decreased alveolar-arterial O2 differences, increased compliance, and decreased Kf values as compared to both EC and UW. CONCLUSIONS: Celsior provides better lung preservation than EC or UW as demonstrated by increased oxygenation, decreased capillary permeability, and improved lung compliance, particularly at 12-hr storage times. These results are highly relevant, inasmuch as EC and UW are the most common clinically used lung preservation solutions. Further studies of Celsior in experimental and clinical lung transplantation, as well as in other solid organs, are indicated.     

Clinical feasibility of a free-weight strength-training program for older adults

In the present study the intergraft mRNA formation immediately before and after transplantation of human livers was investigated by semi-quantitative polymerase chain reaction. The analysis was carried out with mRNA isolated from biopsies routinely taken perioperatively and included the determination of the expression of tumour necrosis factor alpha (TNF-alpha), interleukin (IL-6), IL-8, IL-10, transforming growth factor beta (TGF-beta) and beta-actin. It was found that biopsies obtained 30-60 min after reperfusion of the liver graft contained significantly higher levels of mRNA for TNF-alpha, IL-6 and IL-8 than biopsies collected subsequently to cold preservation. No such differences were obtained for TGF-beta and IL-10 mRNA. Considerable interindivdual differences were observed concerning the degree of inducibility, in particular for IL-6 mRNA. Retrospective comparison with the clinical course of the individual patients revealed a close and statistically significant correlation between low IL-6 expression and the occurrence of acute rejection episodes within 30 postoperative days, while high IL-6 mRNA levels coincided with the absence of rejection signs. High values for TNF-alpha mRNA were associated with ensuing acute rejection episodes.     

Endothelin-1 levels in ischaemia, reperfusion, and haemorrhagic shock in the canine infrarenal aortic revascularisation model

Endothelin-1 (ET-1) is a potent vasoconstrictive polypeptide produced from vascular endothelial cells. The effects of ischaemia, reperfusion, and exsanguination on plasma ET-1 levels were studied and compared in the mongrel dog after infrarenal aortic cross clamping. Ischaemia produced a trend toward increased ET-1 serum levels (p < 0.07 with Bonferroni correction) that did not reach significance. Plasma ET-1 levels were significantly increased during reperfusion and even further elevations were found following exsanguination. We found a 2-3 fold increase in ET-1 levels following reperfusion (Initial 3.19 +/- 0.27 pg/ml vs. Reperfusion maximum 6.32 +/- 0.72 pg/ml, Bonferroni p < 0.01). Haemorrhagic shock was associated with a 3-4 fold increase in ET-1 levels (Initial 3.19 +/- 0.27 pg/ml vs. Exsanguination maximum 8.37 +/- 0.97 pg/ml Bonferroni p < 0.001). These data reveal that ET-1 is released during reperfusion and exsanguination and may mediate remote vascular events associated with infrarenal aortic cross clamping and acute blood loss.