A study of the 30 minutes following reperfusion after crystalloid and cold blood cardioplegia by enzymatic and metabolic analysis of coronary blood flow

Post-ischemic reperfusion phenomena were studied in two methods of myocardial protection: crystalloid cardioplegia (St Thomas n(o) 2) and cold blood cardioplegia (Buckherg) during cardiopulmonary bypass for human myocardial revascularisation. Myocardial protection was assessed on the course of hemodynamic parameters, reperfusion arrhythmias and biochemical analysis of the coronary flow after cross-clamp removal: creatine phosphokinase (CPK-MB) and nucleotide adenine metabolites (adenosine, inosine, hypoxanthine, xanthine and uric acid). The study was performed in two groups of 14 patients. Hemodynamic conditions were similar in both groups during reperfusion in order to avoid different coronary flow. Under these conditions, myocardial protection by cold blood cardioplegia reduced reperfusion arrhythmias, and resulted in a loss of CPK-MB release. Furthermore, cold blood cardioplegia provided protection of myocardial energy metabolism by reducing the loss of metabolites, purine bases and oxypurine bases into the coronary sinus. Our results also show that hypoxanthine is probably the final product of ATP degradation in human myocardial tissue.     

The role of xanthine dehydrogenase (xanthine oxidase) in ischemia-reperfusion injury in rat kidney

Xanthine dehydrogenase (XDH) and xanthine oxidase (XO) are enzymes involved in the metabolism of purines in various organisms. XO produces superoxide radicals, suggesting that is responsible for tissue ischemia-reperfusion injury. To test this notion further studies were performed on rat kidneys and the time course of changes in purine nucleotides, oxypurines and XDH and XO activity was determined. At 24 hours after reperfusion subsequent to 30-minute ischemia, serum creatinine increased to 0.83 +/- 0.74 mg/dl from 0.28 +/- 0.06 mg/dl (the level prior to ischemia, the control). Renal ATP and ADP contents were reduced after ischemia lasting for 30 minutes and restored 10 minutes after reperfusion following 30 minutes of ischemia. The renal AMP content increased after 30 minutes of ischemia and recovered within 10 minutes after reperfusion. The total adenine nucleotide (TAN) content was reduced gradually during ischemia-reperfusion in the rat kidney. Although the energy charge was reduced following 30 minutes of ischemia, it was restored to the control level 10 minutes following reperfusion. Hypoxanthine (HX) and xanthine (X), which had accumulated at 30 minutes after ischemia, were reduced to the control levels 10 minutes after reperfusion. There were no significant changes in the pre-ischemia values of total XDH and XO activities or XDH/XO ratio during the period nor at various time intervals (up to 24 hours) during reperfusion. It was shown that HX and X accumulate without significant conversion of XDH to XO during ischemia. Therefore the putative role of XO in ischemia-reperfusion injury seems to more complex than initially predicted.     

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.     

Hepatic energy charge and adenine nucleotide status in rats anesthetized with halothane, isoflurane or enflurane

BACKGROUND: Volatile anesthetics are known to have varying effects on hepatic oxygen supply in vivo and have been shown to depress hepatic mitochondrial respiration and so energy charge in vitro. However, the effect of halothane, isoflurane and enflurane on hepatic adenine nucleotide status in vivo has not been evaluated. METHODS: Ninety male rats were exposed to 40% oxygen (n = 22) or 40% oxygen in equipotent (1 MAC) concentrations of halothane (1%) (n = 23), isoflurane (1.4%) (n = 22) or enflurane (2%) (n = 23) for 2 hours. All animals were then administered intraperitoneal pentobarbital and anesthesia continued and laparotomy was performed. A liver biopsy was taken for determination of hepatocellular adenosine-5-triphosphate (ATP), adenosine-5-diphosphate (ADP) and adenosine-5-monophosphate (AMP) and computation of energy charge (EC) from ?(ATP + 1/2ADP)+(ATP + ADP + AMP)? and total adenine nucleotides (TAN) from (ATP + ADP + AMP). After the biopsy the aorta was cannulated for blood sampling. RESULTS: Rats in each group were similar in weight, as well as acid base and blood gas status just after liver biopsy. Hepatic energy charge, ATP, ADP, AMP, and TAN levels were not different in animals receiving either halothane, isoflurane or enflurane when compared with those receiving only oxygen. CONCLUSION: One MAC of anesthesia for a period of 2 hours with the described volatile anesthetic agents did not affect adenine nucleotide status in vivo in rats.     

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.     

Myocardial uptake of 99mTc-tetrofosmin, sestamibi, and 201Tl in a model of acute coronary reperfusion

BACKGROUND: To investigate whether tetrofosmin uptake is affected by myocardial viability as has been noted for 201Tl and sestamibi, we analyzed the initial and delayed distribution patterns of tetrofosmin in a rat coronary artery occlusion-reperfusion model. METHODS AND RESULTS: Animals were intubated and ventilated, and their arterial pressures were monitored. A left thoracotomy was performed. After 1-hour occlusion and 1-hour reperfusion of a major branch of the circumflex artery, 201Tl and either tetrofosmin or sestamibi were injected intravenously. Radiolabeled microspheres were used to document the area at risk and reperfusion. Five minutes or 1 hour after administration of the diffusible tracers, the animals were killed. Tracer distribution was determined by use of segmental tissue analysis, and tissue viability was determined by use of histochemical staining. Both the initial and delayed retention of tetrofosmin were sensitive to myocardial viability, as shown by significantly lower uptake (30 +/- 14%) and retention (24 +/- 12%) of tetrofosmin in the nonviable segments compared with the viable segments. In addition, the initial myocardial distribution of tetrofosmin was similar to that noted for 201Tl, but after 1 hour of tracer circulation, the tetrofosmin tissue distribution appeared unchanged compared with the initial regional blood flow distribution. This is in direct contrast to our present observations of significant 201Tl redistribution and some changes in sestamibi distribution as well. CONCLUSIONS: The clinical implication of these observations suggests that initial and delayed imaging after tetrofosmin administration would reflect both the initial regional blood flow pattern and myocardial viability.     

Nasal T/natural killer (NK)-cell lymphomas are derived from Epstein-Barr virus-infected cytotoxic lymphocytes of both NK- and T-cell lineage

OBJECTIVES: Previous studies have reported that hyperinflation during lung ischemia improves pulmonary function after reperfusion. However, it has not been clarified whether hyperinflation itself or oxygen in inflation gas causes good pulmonary function. The aim of this study is to evaluate the effect of oxygen in pulmonary inflation gas during lung ischemia on ischemia-reperfusion injury. METHODS: Twenty-one mongrel dogs were randomly divided into three groups: the lung during a 90-minute period of warm ischemia was inflated to 30 cm H2O with 100% oxygen in group A and 100% nitrogen in group B; it was not inflated in group C. Pulmonary function and hemodynamics were measured before ischemia and 1, 2, and 3 hours after reperfusion. Total protein and phosphorus of phospholipid in bronchoalveolar lavage fluid were measured 210 minutes after reperfusion. Adenine nucleotide levels in lung tissue were estimated 210 minutes after reperfusion. RESULTS: No significant differences in pulmonary function and hemodynamics were noted between group A and group B, but these two groups had significantly better pulmonary function and hemodynamics than group C. No significant differences were detected in the concentrations of total protein and phosphorus of phospholipids in bronchoalveolar lavage fluid and in adenine nucleotide levels of lung tissue after reperfusion among the three groups. CONCLUSIONS: The results indicate that pulmonary inflation during warm ischemia improves pulmonary function and hemodynamics after reperfusion in this model. The effect is caused by inflation itself and is not due to oxygen as a metabolic substrate during warm ischemia.     

Structure and expression of a murine homologue of sky receptor tyrosine kinase gene

We examined effects of adenine nucleotide on ischemic myocardial stunning in dogs. Pentobarbitalanesthetized open-chest dogs were subjected to 20-min ligation of the left anterior descending coronary artery (LAD), followed by reperfusion for 30 min. Either saline, 5 mM 8-bromo-5'-AMP (tributyryl-AMP), or 30 mM N6, 2', 3'-tributyryl-5'-AMP (tributyryl-AMP), 5 mM 5-amino-4-imidazole carboxamide riboside (AICAr) as a positive reference, was infused at 0.1 ml/kg/min in the left femoral vein throughout the experiment. The myocardial contractile function was measured by ultrasonometry. The tissue levels of high-energy phosphates in the reperfused heart were determined. Myocardial contractile function assessed by % segment shortening (%SS) in the saline-infused group decreased during ischemia and returned toward the preischemic level during reperfusion but incompletely. A significant improvement in the %SS during reperfusion was observed in the 8-bromo-AMP- and AICAr-infused groups but not in the tributyryl-AMP-infused group. The magnitude of the protective effect of the drugs on myocardial contractility during reperfusion was 8-bromo-AMP > AICAr > tributyryl-AMP = saline. Only in the 8-bromo-AMP-infused group were the levels of ATP, ADP, and total adenine nucleotides in the reperfused heart significantly higher than those in the saline-infused group. The present result indicates that 8-bromo-AMP improves the ability of the heart to recover from ischemia and reperfusion associated with a significant restoration of ATP.     

Effects of intermittent reperfusion on brain pHi, rCBF, and NADH during rabbit focal cerebral ischemia

BACKGROUND AND PURPOSE: The use of intermittent reperfusion versus straight occlusion during neurovascular procedures is controversial. This experiment studied the effects of intermittent reperfusion and single occlusion on intracellular brain pH (pHi), regional cerebral or cortical blood flow, and nicotinamide adenine dinucleotide (NADH) fluorescence during temporary focal ischemia. METHODS: Twenty fasted rabbits under 1.0% halothane anesthesia were divided into four groups: (1) nonischemic controls, (2) 60 minutes of uninterrupted focal ischemia, (3) 2 x 30-minute periods of focal ischemia separated by a 5-minute reperfusion, and (4) 4 x 15-minute periods of focal ischemia separated by three 5-minute reperfusion periods. Focal ischemia was produced by occlusion of both the middle cerebral and ipsilateral anterior cerebral arteries. After the final occlusion, there was a 3-hour reperfusion period in all groups. Regional cerebral and cortical blood flow, brain pHi, and NADH fluorescence were measured with in vivo panoramic fluorescence imaging. RESULTS: During occlusion, regional cerebral and cortical blood flows and NADH fluorescence values were not different among the groups. Brain pHi was significantly lower in the 4 x 15-minute group compared with the 1 x 60-minute group (6.57 +/- 0.02 versus 6.73 +/- 0.06; P < .03) but not significant when compared with the 2 x 30-minute group. During the short reperfusion periods, all parameters returned to normal except for NADH fluorescence levels, which remained elevated. During the postischemic final reperfusion period, there was a mild brain alkalosis of approximately 7.1 in all groups. There were no significant differences in NADH fluorescence among groups during the final reperfusion. Regional cerebral and cortical blood flow returned to near normal values in all groups. CONCLUSIONS: This study demonstrates that intermittent reperfusion during temporary focal ischemia has different effects on the intracytoplasmic and the intramitochondrial compartments: worsening of brain cytoplasmic pHi but no significant differences in the oxidation/reduction level of mitochondrial NADH.     

Midazolam-flumazenil topical anaesthesia for microlaryngoscopy with jet ventilation

BACKGROUND: Enhanced production of endothelin-1 (ET-1), vasoconstrictive 21 amino acids produced by endothelial cells during ischemia and after reperfusion of the liver, is known to cause sinusoidal constriction and microcirculatory disturbances, which lead to severe tissue damage. Using a 2-hour hepatic vascular exclusion model in dogs, we tested our hypothesis that neutralization of ET-1 by monoclonal anti-ET-1 and anti-ET-2 antibody (AwETN40) abates vascular dysfunction and ameliorates ischemia/reperfusion injury of the liver. STUDY DESIGN: After skeletonization, the liver was made totally ischemic by cross-clamping the portal vein, the hepatic artery, and the vena cava (above and below the liver). Veno-venous bypass was used to decompress splanchnic and inferior systemic congestion. AwETN40, 5 mg/kg, was administered intravenously 10 minutes before ischemia (treatment group, n = 5). Nontreated animals were used as controls (control group, n = 10). Animal survival, hepatic tissue blood flow, liver function tests, total bile acid, high-energy phosphate, ET-1 levels, and liver histopathology were studied. RESULTS: Treatment with AwETN40 improved 2-week animal survival from 30% to 100%. Hepatic tissue blood flow after reperfusion was significantly higher in the treatment group. The treatment significantly attenuated liver enzyme release, total bile acid, and changes in adenine nucleotides. Immunoreactive ET-1 levels in the hepatic venous blood of the control group showed a significant increase and remained high for up to 24 hours after reperfusion. Histopathologic alterations were significantly lessened in the treatment group. CONCLUSIONS: These results indicate that ET-1 is involved in ischemia/reperfusion injury of the liver, which can be ameliorated by the monoclonal anti-ET-1 and anti-ET-2 antibody AwETN40.     

Evaluation of early graft function by hepatic venous hemoglobin oxygen saturation following orthotopic liver transplantation in the rat

This study was designed to evaluate the use of hepatic venous oxygen saturation (Shvo2) as a predictor of early graft function after liver transplantation. We examined the levels of Shvo2 and serum ALT after transplantation using the isogeneic rat orthotopic liver transplant model. Shvo2 levels 2 hr after reperfusion in the 6-hr cold ischemia (nonviable allograft) group were significantly lower than those in the 1-hr and 3-hr cold ischemia (viable allograft) groups. However, there was no significant difference in ALT levels among these groups. These results suggest that decreased hepatic blood flow due to microcirculatory disturbances may occur in the nonviable allograft even in the early phase of reperfusion and may be responsible for ischemic damage to parenchymal cells. Therefore, Shvo2 could provide a simple index of the initial graft status and be useful for a rapid etiological diagnosis of early postoperative graft dysfunction and for estimating the graft outcome after liver transplantation.     

Cardioprotection by orotic acid: metabolism and mechanism of action

The pyrimidine base, orotic acid (OA), improves the function of recently infarcted hearts subjected to global ischemia but its mechanism of action is unclear. Our aims were to examine (i) in normal rats, the effect of OA on pyrimidine levels in plasma, liver and heart; (ii) in rats with normal or infarcted hearts, the effect of OA on adenine nucleotide metabolism and mechanical function, before and after global ischemia. To investigate the metabolism of OA, normal rats received 100 mg/kg OA, and changes in plasma and tissue concentrations of pyrimidines were examined. The effects of OA were also studied in rats receiving OA for 2 days after coronary ligation or sham operations, and plasma and tissue pyrimidine concentrations examined. Their hearts were isolated and perfused, then subjected to 30 min of global ischemia. Mechanical function and adenine nucleotide content were assessed pre- and post-ischemia. In normal, unoperated rats, administration of 100 mg/kg OA significantly increased hepatic uracil and cytosine nucleotide concentrations, then increased plasma uridine (+124%) and cytidine (+55%), and transiently increased myocardial uracil nucleotides (+21%). Infarction significantly reduced recovery of cardiac work after global ischemia (sham=62%; infarct=26%; P<0.05), and OA treatment in infarcted hearts increased post-ischemic work by 192% (P<0.05), but not in shams. Pre-ischemic ATP was reduced in the surviving myocardium of infarcted hearts from 21.7+/-0.8 to 14.7+/-0. 7 micromol/g dry weight (P<0.001) and total adenine nucleotides (TAN) from 30.3+/-0.8 to 22.4+/-1.1 micromol/g dry weight (P<0.001). OA treatment prevented these reductions in infarcted hearts (ATP 20. 7+/-0.5; TAN, 29.1+/-0.6 micromol/g dry weight). We conclude that OA protects the infarcted heart against global ischemia by enhancing hepatic release of pyrimidine nucleosides into the plasma, which then prevent depletion of adenine nucleotides in the surviving myocardium.     

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.     

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.     

Protective effect of preservation of canine pancreas by the two-layer (University of Wisconsin solution/perfluorochemical) method against rewarming ischemic injury during implantation

Rewarming ischemia during implantation severely compromises posttransplant pancreas graft survival because the graft has already been subjected to warm and cold ischemia before implantation. The purpose of this study was to examine whether preservation of the pancreas graft by the two-layer method ameliorates rewarming ischemic injury of the graft during implantation using a canine model. After flushing with cold University of Wisconsin solution (UW), the pancreas grafts were preserved by the two-layer (UW/perfluorochemical [PFC]) method (group 1) or simple cold storage in UW (group 2) for 24 hr and then autotransplanted. In control, the pancreas grafts were flushed out with cold UW and immediately autotransplanted without preservation (group 3). After completion of vascular anastomosis, vascular clamp was not released until 90, 120, or 150 min of rewarming ischemia, including anastomosis time, had elapsed. After 90 min of rewarming ischemia, graft survival rates were 5/5, 100%, 5/5, 100%, and 5/5, 100%, in groups 1, 2, and 3, respectively. After 120 min, all the grafts in groups 2 and 3 failed (0/5, 0%, and 0/5, 0%, respectively); however, all the grafts in group 1 survived (5/5, 100%). Even after 150 min, 1 of 3 grafts in group 1 survived (1/3, 33%). After 24 hr preservation, tissue ATP levels of the grafts in group 1 were about 2-fold the reference values before harvesting (8.23 +/- 0.72 vs. 4.44 +/- 0.49 mumol/g dry weight, P < 0.05) and significantly higher compared with group 2 (8.23 +/- 0.72 vs. 1.76 +/- 0.52 mumol/g dry weight, P < 0.01). After 120 min of rewarming ischemia, tissue ATP levels in group 1 were 84% of the reference values and significantly higher compared with group 2 (3.75 +/- 0.25 vs. 1.57 +/- 0.48 mumol/g dry weight, P < 0.05). Two hours after reperfusion, ATP levels in group 1 were 42% of reference values but significantly higher compared with group 2 (1.86 +/- 0.36 vs. 1.03 +/- 0.18 mumol/g dry weight, P < 0.05). We conclude that the two-layer (UW/PFC) method ameliorates rewarming ischemic injury of the pancreas graft during implantation by increasing tissue ATP contents during preservation and consequently maintaining tissue ATP levels during implantation.     

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.     

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.     

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.     

Coronary vascular regulation during postcardioplegia reperfusion

BACKGROUND: This study extends previous investigations of global and regional myocardial blood flow during early postcardioplegia reperfusion. The hypothesis tested is that coronary vascular regulation becomes abnormal within 3 minutes after the start of postcardioplegia reperfusion. METHODS: Pigs (n = 40) were supported by cardiopulmonary bypass and 38 degrees C blood cardioplegic solution was infused. A control preischemic microsphere injection (No. 1) was given in asystolic hearts. Groups 1 to 3 had 1 hour of hypothermic cardioplegic arrest. Group 4 (control group) had 1 hour of perfusion without cardioplegia. A blood cardioplegic solution at 38 degrees C and 70 mm Hg pressure was infused to maintain asystole during the initial 7 to 10 minutes of reperfusion in all groups. Left ventricular intracavitary pressures were set at 0, 10, 20, or 0 mm Hg in groups 1, 2, 3, and 4 (n = 10 pigs per group), respectively, during the initial 7 minutes of reperfusion. The ventricle was then decompressed. At 30 seconds, 3 minutes, and 6 minutes after reperfusion, microsphere injections 2, 3, and 4 were given in asystolic hearts. Microsphere injection No. 5 was given 10 minutes after reperfusion in beating vented hearts. RESULTS: (1) Left ventricular distention during the initial 7 minutes of reperfusion after hypothermic cardioplegic arrest attenuates postischemic hyperemia. (2) Left ventricular intracavitary pressure of 20 mm Hg during reperfusion causes a decrease in endocardial blood flow relative to epicardial blood flow at 6 minutes after reperfusion. (3) Global myocardial blood flow during postcardioplegia reperfusion falls significantly below preischemic control values despite the return of electromechanical activity. INFERENCE: Coronary vascular regulation (i.e., coronary resistance and metabolic flow recruitment) becomes abnormal within 3 minutes after the start of reperfusion after hypothermic blood cardioplegic arrest.     

New insights into positional alcohol nystagmus using three-dimensional eye-movement analysis

BACKGROUND: It is generally accepted that 0 to 4 degreesC is a suitable temperature for organ preservation. The reason for this is based on the premise that at temperatures below 0 degreesC, intracellular ice is likely to form, with subsequent damage to cellular structures. However, it cannot be assumed that subzero temperatures will freeze the cell. In this study, we attempted to confirm the specific freezing point of rat liver and to preserve it at a temperature just above that point. METHODS: Rat livers were stored for 24, 48, 72, and 96 h either at 4 degreesC (Group N) or at -0.8 degreesC (just above the temperature ascertained to be the specific freezing point of rat liver; Group H). After cold storage, the livers were perfused for 60 min using an isolated perfused liver model for assessment of liver function. RESULTS: ATP and TAN (total adenine nucleotides) in reperfused liver tissues were significantly higher in Group H than in Group N for all preservation periods. ADP was significantly higher in Group H than in Group N for 24-, 72-, and 96-h preservation periods. Energy charge was significantly higher in Group H than in Group N for 24-, 48-, and 96-h preservation periods. CONCLUSIONS: Regarding the content of ATP, ADP, and TAN and the adenylate energy charge, our results indicate that preservation at -0.8 degreesC is advantageous. This novel preservation technique seems to prolong the period that organs can be stored.