Extending the margin of safety of preservation period for resuscitation of ischemically damaged pancreas during preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C with thromboxane A2 synthesis inhibitor OKY046

We have shown that 5-hr preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C allows ATP synthesis and makes it possible to resuscitate a canine pancreas subjected to 90 min of warm ischemia. However, 8 hr of preservation using this method caused a disturbance of vascular microcirculation and did not resuscitate the grafts. The aim of this study was to examine the effect of thromboxane A2 synthesis inhibitor OKY046 on vascular endothelial cells and ATP tissue levels of canine pancreas during preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C, and vascular microcirculation and pancreas viability after transplantation. Graft viability was judged by graft survival following autotransplantation. ATP tissue levels were measured by high-performance liquid chromatography at the end of preservation. Viability of the vascular endothelial cells was judged using nuclear trypan blue uptake of the graft after preservation. Pancreatic tissue perfusion was measured using an H2 clearance technique after reperfusion. Pancreas grafts subjected to 90 min of warm ischemia were not viable (0/5). However, 5-hr preservation made it possible to recover the pancreas (5/5); 8-hr preservation was not successful (0/3). ATP tissue levels after 5-hr and 8-hr preservation were 9.40+/-2.09 and 7.37+/-1.06 micromol/g dry weight, respectively, and OKY046 did not affect ATP synthesis during 8-hr preservation (8.44+/-0.92 micromol/g dry weight). The percentage of nuclear trypan blue uptake of endothelial cells in 8-hr-preserved grafts was 37.6+/-11.6% and was significantly higher than the value in 5-hr-preserved grafts (5.0+/-3.0%; P<0.01). However, OKY046 significantly reduced trypan blue uptake in 8-hr-preserved grafts (8.2+/-3.6%; P<0.01). Pancreatic tissue perfusion in 8-hr-preserved grafts after 2 hr of reperfusion was 28.5+/-7.5 ml/min/100 g, and was significantly lower than the value in 5-hr-preserved grafts (57.1+/-4.4 ml/ min/100 g; P<0.01), but OKY046 dramatically improved pancreatic tissue perfusion (97.1+/-14.6 ml/min/100 g; P<0.01). As a consequence, 8-hr-preserved grafts were resuscitated (4/5). We conclude that OKY046 protects the vascular endothelium during preservation by the two-layer method at 20 degrees C and consequently improves vascular microcirculation on reperfusion. Together with ATP synthesis, which is essential for repairing damaged cells, the canine pancreas graft subjected to 90 min of warm ischemia is resuscitated during 8-hr preservation by the two-layer method at 20 degrees C. This method holds promise for pancreas-kidney transplantation from cardiac arrest donors.     

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.     

Excellence of the two-layer method (University of Wisconsin solution/perfluorochemical) in pancreas preservation before islet isolation

BACKGROUND: In islet transplantation pancreatic preservation before islet isolation is an obstacle compromising islet yield and viability. We tested the feasibility of a two-layer method (University of Wisconsin solution [UW]/perfluorochemical) for pancreatic preservation before islet isolation. METHODS: Dog pancreases were processed into pure islets by the method of Ricordi preceded by five different preservations (groups 1-a and 1-b, the two-layer method for 3 and 24 hours; groups 2-a and 2-b, simple cold storage in UW for 3 and 24 hours; group 3, without preservation). Islet yields and functional success after autotransplantation into the liver were compared among the groups. RESULTS: Postpurification islet equivalents (IE)/gm pancreas and functional success rate were 5600 (mean), 83% in group 1-a; 4000, 56% in group 1-b; 4700, 33% in group 2-a; 1300, 0% in group 2-b; and 5000, 89% in group 3 (p < 0.05; 2b versus 1-a, 1-b, and 3), respectively. There was no statistical difference among groups 1-a, 1-b, and 3 in terms of islet yield and function (p > 0.2). CONCLUSIONS: The two-layer method is more effective than conventional simple cold storage in UW for pancreatic preservation before islet isolation. Clinical trials with the two-layer method are warranted.     

Intracellular volumes and membrane permeability in rat hearts during prolonged hypothermic preservation with St Thomas and University of Wisconsin solutions

The study aims to determine a possible relationship between intracellular water, energy metabolism, functional recovery and membrane permeability, during and after hypothermic cardiac preservation. Isolated rat hearts were stored for 12 h at 4 degrees C with University of Wisconsin (UW), St Thomas Hospital (ST) and Krebs-Henseleit (KH) solutions, and were reperfused for 1 h. Cellular volumes were measured by 1H NMR of water and 59Co NMR of the extracellular marker cobalticyanide, and energetic profiles by 31P NMR spectroscopy. Storage in ST solution reduced ischemic swelling from 2.50 +/- 0.06 to 2.73 +/- 0.09 (P < 0.001 v 3.56 +/- 0.10 ml/g dry weight in KH), while UW solution caused cellular shrinkage to 2.12 +/- 0.08 ml/g dry weight. Intracellular ATP concentrations and pH values were higher in UW as compared to ST solution. At reperfusion, hearts stored in ST shrank while those stored in UW expanded, resulting in similar intracellular volumes. Storage with UW was superior to ST in post-ischemic function 65 +/- 5% (P < 0.01 v 49 +/- 4% with ST) and in recovery of ATP 46 +/- 3% (P < 0.001 v 25 +/- 4% with ST). Storage with both ST and UW solutions did not prevent interstitial edema. Sarcolemmal membrane integrity, as assessed by cellular swelling in response to a hypo-osmotic shock (210 mmol/l), was significantly improved by ST and UW solutions as compared to KH (P < 0.05). Creatine kinase efflux was reduced by ST and UW as compared to KH (P < 0.05), and by UW as compared to ST (P < 0.05). Coronary flow was higher following storage with UW than ST solutions. 66 +/- 6 and 45 +/- 4%, respectively (P < 0.01). According to these data, the beneficial effects of UW and ST solutions on hypothermic ischemic storage of rat hearts included prevention of cellular edema and preservation of sarcolemmal membrane integrity. It is concluded: (a) UW and ST solutions reduce ischemic and reperfusion cellular volumes: (b) both solutions, and UW in particular were efficient in preservation of membrane integrity: (c) prevention of cellular edema is not the single or main mechanism responsible for the improved preservation with UW and ST solutions.     

Intermittent ischemia: energy metabolism, cellular volume regulation, adenosine and insights into preconditioning

Interruption of ischemia by brief reperfusions (I/R) is better tolerated by the heart than continuous ischemia. The present study aims to determine the metabolic profiles of isolated rat hearts during intermittent ischemia, the possible cardioprotective role of adenosine and the influence of I/R on intracellular volumes, using multinuclear NMR spectroscopy. After five I/R (5/5 min) episodes, hearts paced at 5 Hz developed pressures comparable to those of hearts continuously perfused for 50 min at 37 degrees C (CP). Following the first 5 min episode of no-flow ischemia, [ADP] dropped from 72 +/- 9 to 43 +/- 5 microM (P < 0.001) and remained stable at the end of the following reperfusions, despite a 2.5-4-fold increase during each episode of 5 min ischemia. Intracellular volumes were stable during CP at a value of 2.50 +/- 0.06 ml/g dry weight, and decreased by 4, 8, and 12% after 1, 3, and 5 I/R episodes. The phosphorylation potentials decreased from 54 +/- 8 to 4 mM-1 during each period of 5 min ischemia and were 40 +/- 6 and 28 +/- 6 mM-1 after CP and I/R5, respectively. Cardiac glycogen had decreased during 50 min of CP from 103 +/- 13 to 81 +/- 9 mumol/g dry weight and lactate production was 116 +/- 15 mumol/heart. Five I/R episodes decreased glycogen to 46 +/- 7 mumol/g dry weight (P < 0.005 v CP) and increased lactate efflux to 262 +/- 31 mumol/ heart (P < 0.005 v CP). These findings suggest that a brief ischemia/reperfusion episode increases anaerobic metabolism of exogenous glucose, reduces [ADP] and induces cellular shrinkage. Administration of the adenosine receptor blocker 8-phenyl theophylline (8PT) during intermittent perfusion depressed the developed pressure to 78 +/- 7%, accentuated the decrease in phosphorylation potential (14 +/- 4 mM-1), abolished cellular shrinkage, reduced lactate efflux and blunted the decrease in ADP following the first I/R episode. In variance, no detectable changes were observed during intermittent ischemia when the ATP-sensitive potassium channel blocker glibenclamide was administered. These data demonstrate: (a) a brief episode of ischemia/reperfusion stimulates anaerobic metabolism of exogenous glucose and lowers intracellular ADP concentration: (b) adenosine receptors are partially responsible for the glycolytic stimulation during intermittent ischemia; (c) cellular shrinkage is related to the rate of glycolysis during intermittent ischemia/reperfusion.     

Ischaemia-reperfusion injury of the liver: treatment in theory and in practice

Cold ischaemia-reperfusion injuries are an unavoidable feature of current liver transplantation procedures. Damage to liver grafts accures mainly from hypothermic storage under hypoxic conditions (cold ischaemia), from sustained ischaemia during implantation into the recipient (rewarming ischaemia) and from restoration of blood and oxygen to the graft (reperfusion injury). These three stages are characterized by progressive deteriorations in hepatic function, with sinusoidal endothelial cells most affected during cold ischaemia. Activation of Kupffer cells (hepatic macrophages) at reperfusion augments damage to both endothelial and parenchymal cells by the release of numerous compounds which initiate and perpetrate injury and impair the hepatic microcirculation. The key events in the expression of ischaemia-reperfusion injury are detailed and therapeutic interventions are described which target these steps. The treatments discussed include University of Wisconsin (UW) preservation solution, calcium channel blockade, inhibitors of Kupffer cell activation, promoters of microvascular vasodilation, hepatoprotectants and the use of anti-oxidants.     

The effect of coenzyme Q10 and cold cristalloid cardioplegia on hypothermic global ischemia

Coenzyme Q10 (CoQ10, ubiquinone) has been shown to be protective against myocardial ischemia/reperfusion induced injury. The purpose of this study was to investigate the effect of CoQ10 added to cold cristalloid cardioplegia on hypothermic ischemia and normothermic reperfusion using an isolated working rat heart. Hearts (n = 6-9/group) from male Wistar rats were aerobically (37 degrees C) perfused (20 min) with bicarbonate buffer. This was followed by a 3-min infusion of St. Thomas' Hospital cardioplegic solution containing various concentrations of CoQ10 (0, 1, 3, 6, 12, and 58 mumol/L). Hearts were then subjected to 180 min of hypothermic (20 degrees C) global ischemia and 35 min of normothermic (37 degrees C) reperfusion (15 min Langendorff, 20 min working). Ventricular fibrillation (Vf) upon reperfusion was irreversible in the 12 and 58 mumol/ L CoQ10 groups (4/6 and 3/6, respectively). In the hearts which Vf upon reperfusion was not irreversible, the percent recovery of aortic flow (%AF) was 43.3 +/- 5.4% (n = 9) in the control group versus 31.6 +/- 7.7% (n = 6), 38.0 +/- 12.0% (n = 6), 27.2 +/- 6.9% (n = 6), 31.3% (n = 2), and 30.4 +/- 14.2% (n = 3) in the 1, 3, 6, 12, and 58 mumol/L CoQ10 groups, respectively. Creatine kinase leakage during Langendorff reperfusion tended to be greater in the 12 and 58 mumol/L CoQ10 groups than in the control group. Thus, CoQ10 in the cold cristalloid cardioplegic solution induced irreversible Vf upon reperfusion and failed to improve functional recoveries following hypothermic global ischemia.     

Changes in glucose transporter 2 and carbohydrate-metabolizing enzymes in the liver during cold preservation and warm ischemia

In order to examine glucose metabolism in liver grafts during cold preservation (24 and 48 hr), warm ischemia (60 and 120 min), a combination of the two and reperfusion, the amount of protein and mRNA of glucose transporter 2 and the activities of enzymes in glycolysis (glucokinase, phosphofructokinase, pyruvatekinase), gluconeogenesis (glucose 6-phosphatase, fructose 1,6-bisphosphatase), and the pentose phosphate pathway (glucose 6-phosphate dehydrogenase) were measured. It appeared that glucose transport, the pentose phosphate pathway, and gluconeogenesis were maintained during cold preservation and warm ischemia. The activity of glucokinase significantly decreased from the control value of 1.33 +/- 0.23 IU/g protein to 0.70 +/- 0.17 (24 hr, P<0.05) and 0.57 +/- 0.12 (48 hr, P<0.01) only during cold preservation. However, the activity of phosphofructokinase significantly decreased from the control value of 4.37 +/- 0.06 IU/g protein to 2.67 +/- 0.15 (60 min, P<0.0001) and 1.53 +/- 0.06 (120 min, P<0.0001) only during warm ischemia. This indicates that glycolysis deteriorates during both cold preservation and warm ischemia and demonstrates further that the balance between glycolysis and gluconeogenesis shifts to gluconeogenesis. Even when cold preservation was combined with warm ischemia, the activity of glucokinase decreased only during cold preservation and the activity of phosphofructokinase decreased only during warm ischemia. Furthermore, these changes were time-dependent. It is suggested that they can be used as a clock to measure the durations of cold preservation and warm ischemia separately and that the magnitude of an ischemic injury to a liver and a liver graft's viability can be indirectly estimated before transplantation.     

Homeobox genes in hematopoiesis and leukemogenesis

The effect of cold and warm intermittent antegrade blood cardioplegia, on the intracellular concentration of taurine in the ischaemic/reperfused heart of patients undergoing aortic valve surgery, was investigated. Intracellular taurine was measured in ventricular biopsies taken before institution of cardiopulmonary bypass, at the end of 30 min of ischaemic arrest and 20 min after reperfusion. There was no significant change in the intracellular concentration of taurine in ventricular biopsies taken after the period of myocardial ischaemia in the two groups of patients (from 10.1 +/- 1.0 to 9.6 +/- 0.9 mumol/g wet weight for cold and from 9.3 +/- 1.3 to 10.0 +/- 1.3 mumol/g wet weight for warm cardioplegia, respectively). Upon reperfusion however, there was a fall in taurine in both groups but was only significant (P < 0.05) in the group receiving cold blood cardioplegia (6.9 +/- 0.8 mumol/g wet weight after cold blood cardioplegia versus 8.0 +/- 0.8 mumol/g wet weight following warm blood cardioplegia). Like taurine, there were no significant changes in the intracellular concentration of ATP after ischaemia in the two groups of patients (from 3.2 +/- 0.32 to 2.95 +/- 0.43 mumol/g wet weight for cold and from 2.75 +/- 0.17 to 2.62 +/- 0.21 mumol/g wet weight for warm cardioplegia, respectively). However upon reperfusion there was a significant fall in ATP in both groups with the extent of the fall being less in the group receiving warm cardioplegia (1.79 +/- 0.19 mumol/g wet weight for cold and 1.98 +/- 0.27 mumol/g wet weight for warm cardioplegia, respectively). This work shows that reperfusion following ischaemic arrest with warm cardioplegia reduces the fall in tissue taurine seen after arrest with cold cardioplegia. Accumulation of intracellular sodium provoked by hypothermia and a fall in ATP, may be responsible for the fall in taurine by way of activating the sodium/taurine symport to efflux taurine.     

Effect of ischemic preconditioning on adenine nucleotide levels of graft lung from canine donor

Twelve canine left lung allotransplantation were performed. In the ischemic preconditioning group (Group IP, n = 6), left donor lung was preconditioned with 10 min ischemia followed by 15 min reperfusion using the occuluding left hilum before resection and cold perfusion. The control group (Group C, n = 6) underwent the same treatment but without ischemic preconditioning. Adenine nucleotides (ATP, ADP, AMP) of the donor lung tissue were measured using rHPLC after 2 hr of resection and cold perfusion with Euro-Collins solution (ECS). The results showed that contents of ATP and total adenine nucleotides (TAN) were much higher in Group IP than in Group C (322.9 +/- 61.2, 942.9 +/- 134.5 and 200.0 +/- 50.0, 668.4 +/- 59.6 mumol.g-1 respectively, P < 0.05). Histologic examination of the donor lung in Group IP showed less damage than in Group C after 2 hr of transplantation. The results suggest that IP combined with cold ECS perfusion can reduce the energy metabolism in canine donor lung.     

Energy metabolism and tissue blood flow as parameters for the assessment of graft viability in rat small bowel transplantation

The assessment of small bowel graft viability by means of energy metabolism and tissue blood flow was investigated and compared with pathological findings. Syngeneic heterotopic small bowel transplantations were performed using male Lewis rats, which were divided into four groups according to the duration of cold preservation in University of Wisconsin (UW) solution; 6-, 12-, 24-, and 48-hour groups. The adenine nucleotide metabolism, the tissue blood flow, and the pathological profiles of the grafts were all compared among the groups. The adenosine triphosphate (ATP) levels at the end of cold storage and at 30 minutes after reperfusion, as well as the total adenine nucleotide (TAN) levels at the end of cold storage, before reperfusion, and at 30 minutes after reperfusion were significantly lower in the 48-hour group than those in the other groups, and the blood flow level at reperfusion was significantly lower in the 48-hour group than that in the others. Histological damage after reperfusion extended deep into the crypt layer in the 48-hour group but was confined to within villi in the other groups. These results suggest that the tissue ATP, TAN, and the blood flow levels are considered useful parameters for the assessment of small bowel graft viability.     

Pyruvate reverses fatty-acid-induced depression of ventricular function and calcium overload after hypothermia in guinea pig hearts

OBJECTIVE: High levels of free fatty acids have been shown to impair mechanical recovery and calcium homeostasis of isolated rat hearts following hypothermic perfusion. The objective of the present study was to investigate whether inhibition of fatty acid oxidation through activation of pyruvate dehydrogenase by millimolar concentrations of pyruvate could influence functional recovery and Ca2+ homeostasis after a hypothermic insult. METHODS: Ventricular function and myocardial calcium ([Ca]total) were measured in 3 different groups of Langendorff-perfused guinea pig hearts exposed to 40 min hypothermic (15 degrees C) perfusion, followed by 30 min rewarming at 37 degrees C. The hearts were perfused with either 11.1 mM glucose (G), glucose and 1.2 mM palmitate (GP), or glucose, palmitate and 5 mM pyruvate (GPP) as energy substrates. RESULTS: All groups showed marked elevations in [Ca]total during hypothermia (from 0.6-0.7 mumol.g dry wt-1 to 9.3-12.2 mumol.g dry wt-1 at 40 min hypothermia, P < 0.05), associated with a pronounced increase in left ventricular end-diastolic pressure (LVEDP from 0-2 to 50-60 mmHg). Following rewarming, GP-perfused hearts showed significantly lower recovery of mechanical function compared to both G- and GPP-perfused hearts (% recovery of left ventricular developed pressure: 27 +/- 8 vs. 62 +/- 3 and 62 +/- 8%, respectively, P < 0.05). The reduced mechanical recovery of GP-perfused hearts was associated with elevated [Ca]total. In separate experiments we found that addition of 1.2 mM palmitate reduced glucose oxidation ([14C]glucose) from 1.77 +/- 0.28 mumol.min-1.g dry wt-1 (G-perfused hearts) to 0.15 +/- 0.04 mumol.min-1.g dry wt-1 (GP-perfused hearts, P < 0.05), implying that fatty acids had become the major substrate for oxidative phosphorylation. Fatty acid oxidation was, however, less pronounced after further addition of 5 mM pyruvate. Thus, palmitate oxidation ([3H]palmitate) was more than 40% lower in GPP-perfused than in GP-perfused hearts (0.83 +/- 0.22 vs. 1.41 +/- 0.12 mumol.min-1.g dry wt-1, P < 0.05). CONCLUSIONS: The present results demonstrate impaired ventricular function and calcium homeostasis after hypothermia in guinea pig hearts perfused with fatty acids in addition to glucose, as compared to hearts perfused with glucose alone. Furthermore, we show that these unfavourable effects of fatty acids can be overcome by an exogenous supply of pyruvate.     

Fas/Apo-1 (CD95) receptor lacking the intracytoplasmic signaling domain protects tumor cells from Fas-mediated apoptosis

Despite improved preservation methods, graft dysfunction after liver transplantation continues to contribute considerably to postoperative morbidity and mortality. In clinical and experimental studies prostaglandin (PG)I2 analogs proved effective in the treatment of liver damage of different origin. Using in vivo fluorescence microscopy in a rat liver transplantation model, we studied the effect of donor bolus pretreatment with the PGI2 analog epoprostenol on hepatic graft revascularization. After epoprostenol bolus pretreatment (group 1: liver transplantation/PGI2), perfusion of liver sinusoids after reperfusion was significantly improved as compared with untreated donor livers (group 2: liver transplantation (95.2+/-0.6% vs. 75.3+/-3.8%, mean +/- SEM; P=0.001) and epoprostenol was found almost in the range of that in normal nontransplanted livers (99.4+/-0.2%). In addition, leukocyte adherence in liver lobules (21.0+/-3.5 vs. 115+/-11.5 n/lobule; P=0.001) and postsinusoidal venules (23.0+/-3.8 vs. 113+/-11.3 n/mm2 endothelial surface; P=0.002) was significantly reduced in the pretreated grafts. Bile production in the recipient was significantly increased by epoprostenol pretreatment of the donor (1.88+/-0.4 vs. 0.63+/-0.13 g/100 g liver*1 hr; P=0.015), indicating restored liver function. These results suggest that the prostacyclin analog epoprostenol is effective in preconditioning the graft prior to transplantation, i.e., improving preservation and increasing graft resistance to ischemia/reperfusion injury. Thus, favorable effects on early graft function after clinical liver transplantation may be achieved by introducing epoprostenol pretreatment into the harvesting procedure.     

Glycolysis and energy metabolism in rat liver during warm and cold ischemia: evidence of an activation of the regulatory enzyme phosphofructokinase

The current study was undertaken so that the effects of both ischemia and ischemia + hypothermia could be examined in mammalian liver. Particular reference was made to the function of glycolysis, which is the only mechanism for energy production under these conditions. The response of adenylate pools reflected the energy imbalance created during warm ischemia within minutes of organ isolation. ATP levels and energy charge values for control (freshly isolated) livers were 1.20 +/- 0.07 and 0.49 +/- 0.02 mumol/g. Within 5 min of warm ischemia, ATP levels had dropped well below control values and by 30 min warm ischemia, ATP, AMP, and E.C. values were 0.21, 2.01, and 0.17 mumol/g, respectively. Cold ischemic livers (flushed with Marshall's citrate solution and stored on ice) exhibited similar, but more protracted, patterns of adenylate depletion (ATP and ADP) and accumulation (AMP). In both warm and cold ischemic livers, levels of fructose-6-phosphate (F6P) and fructose-1,6-bisphosphate (F1,6P2) indicated a marked activation of glycolysis at the phosphofructokinase (PFK) locus after a certain time of ischemia. Although the activations occurred at different times (30 min and 10 h for warm and cold ischemic livers, respectively), the patterns of change in levels of glycolytic metabolites associated with the PFK-catalyzed reaction were similar; levels of F6P dropped and F1,6P2 increased. Changes in metabolite levels (phosphoenol pyruvate and pyruvate) associated with another key suspect regulatory enzyme, pyruvate kinase, indicated no role in regulatory control of glycolysis during warm or cold ischemia. The activation of PFK at 30 min and 10 h of warm and cold ischemia, respectively, may reflect the accumulating effects of loss of intracellular homeostasis, which leads to impending irreversible damage.     

Clinical trial of retrograde warm blood reperfusion versus standard cold topical irrigation of transplanted hearts

BACKGROUND: A prospective, randomized clinical study involving 34 patients undergoing heart transplantation compared myocardial preservation of donor hearts maintained with continuous reperfusion with retrograde warm blood cardioplegia during surgical implantation versus the standard cold topical irrigation. METHODS: Hearts in both groups were arrested with a standard crystalloid solution and maintained in a cold saline solution during transportation. In the retrograde group, cardioplegia was administered through a catheter in the coronary sinus during surgical implantation. An average of 471 +/- 30 mL of hyperkalemic crystalloid solution diluted 1:4 in warm blood from the oxygenator was infused. In the standard group, the heart was kept cold by topical irrigation of cold saline solution and was reperfused only when the ascending aorta was unclamped. RESULTS: Preoperative characteristics of donors and recipients were similar in the two cohorts. Ischemic time average 139 +/- 12 minutes in the retrograde group compared with 130 +/- 11 minutes in the standard group (p = 0.57). Cardiopulmonary bypass time averaged 89 +/- 4 minutes in the retrograde group and 110 +/- 12 minutes in the standard group (p = 0.12). Defibrillation at reperfusion was performed in 4 patients (4/17, 24%) in the retrograde group and 12 patients (12/18, 67%) in the standard group (p = 0.01). There were no deaths in the retrograde group (0/17), whereas in the standard group, 3 patients (3/17) died of early graft failure (p = 0.11). Four early graft failures occurred in the standard group (p = 0.06). Two patients (2/17, 12%) were weaned from bypass with ventricular assist devices in the standard group. The number of subendocardial necrotic cells in the first two weekly endomyocardial biopsy specimens averaged 2.7 +/- 0.8 cells/mm2 in the retrograde group and 5.9 +/- 2.4 cells/mm2 in the standard group (p = 0.12). CONCLUSIONS: Retrograde warm blood reperfusion appears to improve the initial recovery of transplanted hearts. The technique is easy to use and may be a useful approach to graft protection during surgical implantation.     

In vivo cyclin E expression as a marker for early cervical neoplasia

BACKGROUND: Heat shock has been associated with the acquisition of tolerance to a wide variety of stressful conditions, including ischemia. This is partly mediated by the production of various heat shock proteins (HSP), including HSP70. One novel approach to the reduction of ischemia-reperfusion injury after lung transplantation is the induction of HSP70 by heat pretreatment of the donor. The purpose of this study was to investigate the feasibility of this approach in an animal model of lung transplantation. METHODS: Animals were divided into six main groups, with groups I to III representing transplanted animals: In groups I and II, donor animals were anesthetized and then underwent heat stress 6 and 12 hours before organ harvest, respectively. Control animals underwent general anesthesia but no heat stress. After harvest, left lungs from groups I to III were preserved for 18 hours at 40 degrees C and then implanted into isogeneic recipients, which were killed 24 hours after reperfusion to assess graft function. Group IV and V animals underwent heat stress followed by a recovery period of 6 and 12 hours, respectively. Lungs were collected both at the time of harvest (right lungs) and after 18 hours of cold preservation (left lungs). Group VI served as nontransplanted controls. Groups IV to VI did not undergo lung transplantation. RESULTS: At the time of harvest but before implantation, HSP70 was significantly increased in heat-shocked nontransplanted donor lungs (groups IV and V) compared with group VI controls. After 18 hours of cold preservation, HSP70 levels were higher in group IV compared with group V and group VI controls. At 24 hours after reperfusion, mean arterial oxygenation was significantly higher in group I compared with group II and group III controls (290.25+/-24.5 vs 154.5+/-23.9 and 119.6+/-11.3 mm Hg, respectively; P < .001). Myeloperoxidase activity was improved in group I compared with group III controls (0.048+/-0.018 vs 0.137+/-0.036 deltaOD/mg/min, respectively; P < .05). The wet/dry weight ratio was also improved in group I compared with group III controls (6.2+/-0.3 vs. 7.8+/-0.4, respectively; P < .05). CONCLUSIONS: Heat pretreatment of the donor 6 hours before harvest results in increased synthesis of HSP70, which offers a dramatic protective effect against subsequent ischemia-reperfusion injury in the lung isograft.     

The relationship between the adenine nucleotide metabolism and the conversion of the xanthine oxidase enzyme system in ischemia-reperfusion of the rat small intestine

The time course of the energy metabolism after reperfusion, the relationship between the conversion of xanthine dehydrogenase to xanthine oxidase (D-to-O conversion) during ischemia, and the changes of the energy metabolism after reperfusion were studied using an ischemia-reperfusion model in the small intestine of the rat. The rat jejunum underwent an occlusion of the superior mesenteric artery and vein for either 30 minutes (group 1, n = 6) or 90 minutes (group 2, n = 6) with collateral interruption, and then it was reperfused. The contents of the adenine nucleotides in the small intestine of the rat were measured by high-performance liquid chromatography (HPLC) before ischemia, and 30, 60, and 90 minutes of ischemia, as well as 30, 60, 120, and 180 minutes after reperfusion. The recovery level of adenosine triphosphate (ATP) in group 1 (6.05 +/- 0.80 mumol/g dry weight) 30 minutes after reperfusion was significantly higher than that in group 2 (2.28 +/- 1.12 mumol/g dry weight) (P < .001). In addition, the ATP content after reperfusion in group 2 did not change from 30 to 180 minutes after reperfusion. The D-to-O conversion during ischemia in group 1 was not significantly greater than that before ischemia; however, that of group 2 did increase significantly during ischemia (P < .005). These results suggest that the tissue damage from ischemia-reperfusion injury after reperfusion under 90 minutes' ischemia is accomplished within the first 30 minutes after reperfusion. Therefore, the ATP level at 30 minutes after reperfusion may be useful for the evaluation of intestinal viability. Thus, the conversion of the xanthine oxidase enzyme system might play an important role in the expression of ischemia-reperfusion injury.     

Decreased survival in rat liver transplantation with extended cold preservation: role of portal vein clamping time

Primary liver graft dysfunction is currently related to cold ischemia-reperfusion injury, although a wide survival range has been reported using 24-hour preservation in cold University of Wisconsin (UW) solution. We hypothesized that the portal vein clamping time (PVCT) played a more important role than cold preservation injury in the postoperative outcome. Rat liver transplantation was performed using different clamping times after 24-hour cold ischemia in the UW solution. Survival rates, plasma tumor necrosis factor (TNF), and nitrate/nitrite levels were examined. Subsequently, the effect of clamping time was evaluated on hepatocyte and sinusoidal endothelial cell (SEC) function using isolated perfused livers. Survival rate was directly related to clamping time length. Marked increases in TNF and nitrate/nitrite levels were found after surgery, particularly after long clamping times. In perfusion studies, the SEC function was already markedly altered after preservation alone and was not further modified by transplantation. By contrast, the hepatocyte function was moderately altered after transplantation, irrespective of clamping times, even when rats operated with long clamping times were in terminal conditions. In rats, 24-hour preservation in cold UW solution is not a severely compromising condition leading to primary liver nonfunction. Long PVCTs are associated with an endotoxemia-like syndrome more related to a warm intestinal ischemia than to cold ischemia injury of the liver.     

Donor age and graft function

We evaluated survival and renal function of cadaveric donor grafts according to donor age. The median age of the pediatric donors was 7.0 (0.7-16) years in 46 patients [median age 11.8 years (range) 3-16.8 years]. The median age of the adult donors was 34.4 (19-54) years in 59 patients [median age 12.1 years (range) 7-17.3 years]. Thirty patients were treated with azathioprine and prednisolone and 75 with cyclosporine A and prednisolone. The glomerular filtration rate (GFR) and the effective renal plasma flow (ERPF) were determined by the clearances of 51chromium-EDTA and 125iodine-hippurate 1-48 months after kidney transplantation. There was no difference in graft survival between pediatric and adult grafts. There were also no differences in GFR in patients receiving grafts from pediatric or adult donors; 2-3 months after transplantation the GFR in recipients of pediatric grafts was 62 +/- 20 ml/min per 1.73 m2 compared with 61 +/- 21 in those receiving adult grafts. The ERPF in recipients of adult grafts was significantly higher in the 1st month after transplantation: 486 +/- 239 versus 362 +/- 158 ml/min per 1.73 m2. From the 4th to the 6th month after transplantation this difference disappeared: the ERPF of grafts from pediatric donors was 279 +/- 131 ml/min per 1.73 m2 compared with 273 +/- 123 ml/min per 1.73 m2 in grafts from adult donors. Using the single-kidney GFR and ERPF on an age-matched group of probands with minor diseases as references, 2-3 months after transplant the mean GFR of grafts from pediatric donors increased to 118% +/- 51%, whereas the GFR of adult donor grafts fell to 60% +/- 22% over the same period. After 4-6 months the ERPF in pediatric grafts was 96% +/- 55% compared with 50% +/- 22% in adult grafts. We conclude that graft survival and function in children with either a pediatric or an adult graft may not differ because graft function adapts to the requirement of the recipient.     

Influence of preconditioning on rat heart subjected to prolonged cardioplegic arrest

BACKGROUND: Ischemic preconditioning (IP) can reduce lethal injury to the myocardium induced by prolonged ischemia. However, little is known about the effect of preconditioning on the heart subjected to cardioplegic arrest and hypothermic preservation. We evaluated the effect of IP on myocardial metabolism, mechanical performance, and coronary endothelial function after cardioplegic arrest and prolonged hypothermic preservation. METHODS: An isovolumic Langendorff perfused rat heart model was used, and hearts were divided into two groups. The first group (IP, n = 14) was preconditioned by 5 minutes of global normothermic (37 degrees C) ischemia followed by 10 minutes of normothermic reperfusion before 6 hours of cold (4 degrees C) preservation, followed by 60 minutes of reperfusion. The second group (control, n = 15) was subjected to 6 hours of cold preservation followed by 60 minutes of reperfusion without preconditioning. Mechanical function was assessed using left ventricular balloon by constructing pressure-volume curves in two ways: at defined left ventricular volumes or at defined left ventricular end-diastolic pressures. Initially, the volume of the balloon was increased incrementally from 0 to 150 microL in 25-microL steps. Measurements were then repeated with loading balloon to achieve left ventricular end-diastolic pressure of 5, 10, 15, or 20 mm Hg. Myocardial function was assessed before ischemia and at 15 or 60 minutes of reperfusion. Metabolic status of the heart was evaluated by measuring the release of purine catabolites during the initial 15 minutes of reperfusion and concentrations of myocardial nucleotides at the end of reperfusion. Endothelium-mediated vasodilatation was evaluated using 10 mumol/L 5-hydroxytryptamine before and after ischemia. RESULTS: Left ventricular end-diastolic pressure values were significantly lower in the IP group, by 20% to 40%, during the reperfusion phase at each volume of the balloon compared with the control group. The rate-pressure product was more favorable during reperfusion in the IP than in the control group because of a 15% increased heart rate in the IP group. The release of purine catabolites from the heart during the reperfusion phase was reduced (p < 0.01) in the IP group (0.66 +/- 0.04 mumol) relative to the control group (0.92 +/- 0.06 mumol). No difference in the recovery of systolic function, myocardial adenosine triphosphate concentration, or endothelial function was observed between the groups. CONCLUSIONS: Under conditions of cardioplegic arrest and hypothermic preservation, IP can offer additional protection for the heart by preventing an increase in diastolic stiffness. However, metabolic improvement or better preservation of the systolic or endothelial function was not observed in this model.