Enhancement of extended lung preservation with a vasoactive intestinal peptide-enriched University of Wisconsin solution

Vasoactive intestinal peptide (VIP) is a known pulmonary and bronchial vasodilator as well as an oxygen free radical scavenger. Since its effect as an additive to University of Wisconsin (UW) solution for lung preservation has been shown previously, the aim of this study was to determine the ability of VIP to improve lung preservation followed by reperfusion. Four groups of excised Sprague-Dawley rat lungs (n = 24) were studied using an isolated blood perfused working lung model. The first 3 groups of lungs were flushed and stored in UW solution at 4 degrees C for: (1) 4 hr, (2) 18 hr, and (3) 24 hr. Group 4 lungs were flushed with UW solution + VIP (1 microgram/ml) and stored in UW solution + VIP (0.5 microgram/ml) for 24 hr. After preservation, the lungs were reperfused to evaluate their functions for 2 hr or until lung failure occurred (arterial oxygen saturation less than 90% and/or appearance of bronchial fluid in the bronchial cannula). In the lungs stored in UW solution for 24 hr, failure occurred after 10 min of reperfusion and all functions were significantly altered. The addition of VIP to UW solution maintained the functional capacity of the lungs, recorded by lung resistance, lung compliance, elastic work, flow resistive work, shunt fraction, and blood oxygen tension. No statistical difference in these parameters other than shunt fraction was found when the VIP group was compared with the group preserved for 4 hr in UW solution. We conclude that lung preservation can be extended to 24 hr with the maintenance of lung functional capacity if VIP is added to UW solution.     

Trimetazidine prevents renal injury in the isolated perfused pig kidney exposed to prolonged cold ischemia

BACKGROUND: Ischemia caused by cold storage (CS) and reperfusion of the kidney is often responsible for delayed graft function after transplantation. Significant attention has been focused on the cascade of events involved in ischemia-reperfusion injury, with the objective of identifying drugs to ameliorate the functional damage that occurs. METHODS: The purpose of this study was to evaluate the renal function of isolated perfused pig kidneys after 48 hr of CS with Euro-Collins (EC) solution plus trimetazidine (EC+TMZ), standard EC solution, or University of Wisconsin (UW) solution. Normothermic isolated perfused pig kidneys were randomized into five experimental groups: (A) control group (cold flush with cold heparinized saline and immediately reperfused; n=6); (B) cold flush with cold heparinized saline with TMZ (10(-6) M), n=6; (C) 48 hr of CS with EC and reperfusion (n=8); (D) 48 hr of CS with EC+TMZ alone and reperfusion (n=8); (E) 48 hr of CS with UW and reperfusion (n=8). Proton nuclear magnetic resonance spectroscopy and biochemical studies were performed for the functional evaluation during reperfusion. Lipid peroxidation was also determined. Histological examination (optical and electron microscopy) was performed after CS and reperfusion. RESULTS: Using TMZ, the renal perfusate flow rate as well as the glomerular filtration rate and proximal tubular function were significantly improved. This improvement of renal function during reperfusion was correlated with a less significant cellular and interstitial edema. In addition, tubular injury markers were significantly lower in the group preserved with EC+TMZ, and TMZ reduced lipid peroxidation dramatically during reperfusion. CONCLUSIONS: The addition of TMZ to the EC solution increased the preservation quality and renal tubular function, and gave protection from reperfusion injury better than EC alone or UW. These results strongly suggest that TMZ has a cytoprotective effect and may therefore be useful for kidney preservation.     

Rat whole-limb viability after cold immersion using University of Wisconsin and Euro-Collins solutions

The purpose of this study was to compare University of Wisconsin (UW) solution with Euro-Collins (EC) solution in their cold preservation effects on rat limbs. Thirty-six Lewis rat limbs were preserved in EC solution (n=18) or UW solution (n=18) at 4 degrees C for 72 hr, and grafted orthotopically to a syngeneic rat using microsurgical techniques. The surgeon was blinded to the solution used. We evaluated the vascular patency rate and death rate of both groups at day 7 after surgery and performed histological evaluations of bone, muscle, growth plate, and articular cartilage for each specimen of successful grafts in both groups. The vascular patency rates of the EC and UW groups were 27.7% (5/18) and 11% (2/18), respectively, and showed no significant difference. The death rates of the EC and UW groups were 50% (9/18) and 60% (10/18), which were not significantly different. There were no clear differences in histological viability between both groups, in all tissues exclusive of bone marrow and muscle tissue. Our results showed that in comparing EC and UW solutions, one was not significantly superior to the other as a cold immersion storage medium after a 72 hr ischemia-induced reperfusion injury.     

Preservation with 8-bromo-cyclic GMP improves pulmonary function after prolonged ischemia

BACKGROUND: Cyclic guanosine monophosphate (cGMP) is a potent second messenger for the nitric oxide pathway in the pulmonary vasculature. Increased cytosolic cGMP levels elicit pulmonary vasodilatation resulting in decreased pulmonary vascular resistance and maximized pulmonary function after ischemia-reperfusion injury. We hypothesized that the addition of a membrane-permeable cGMP analogue (8-bromo-cGMP) to a Euro-Collins (EC) preservation solution would ameliorate pulmonary reperfusion injury better than prostaglandin E1 injection alone after prolonged hypothermic ischemia. METHODS: All lungs from New Zealand White rabbits (weight, 3 to 3.5 kg) were harvested en bloc, flushed with EC solution, and reperfused with whole blood for 30 minutes. Group 1 lungs (immediate control) were immediately reperfused. Group 2 lungs (control) were stored inflated at 4 degrees C for 18 hours before reperfusion. Groups 3 and 4 lungs were flushed with EC solution containing 200 micromol/L 8-bromo-cGMP and stored at 4 degrees C for 18 and 30 hours, respectively. Fresh, nonrecirculated venous blood was used to determine single-pass pulmonary venous-arterial oxygen gradients at 10-minute intervals. Assays for cGMP, cyclic adenosine monophosphate, nitric oxide synthase activity, and myeloperoxidase were performed on all lung tissue samples. Wet to dry weight ratios were determined after 2 weeks of passive desiccation. RESULTS: Oxygenation (venous-arterial oxygen gradient), pulmonary artery pressure, pulmonary vascular resistance, and edema formation were significantly improved in groups 3 and 4 (addition of 8-bromo-cGMP to EC plus 18 or 30 hours of hypothermic ischemia). Hypothermic storage (groups 2, 3, and 4) decreased both nitric oxide synthase activity and myeloperoxidase levels compared with immediate reperfusion (group 1). CONCLUSIONS: These results suggest that the addition of a membrane-permeable cGMP analogue to an EC pulmonary flush solution improves pulmonary function after prolonged storage compared with EC and prostaglandin (E1) preservation alone. The finding of myeloperoxidase reduced levels after hypothermic storage and subsequent reperfusion may suggest a more important role for pulmonary hemodynamic control in mitigating pulmonary reperfusion injury.     

Nitroglycerin does not alter pulmonary vascular permeability in isolated rabbit lungs

Nitroglycerin (NTG) produces vasodilation by releasing nitric oxide (NO) at the cellular level. Other studies have suggested that NO may directly alter vascular permeability and may alter the development of tissue injury. We therefore examined the effects of NTG on vascular permeability in the buffer-perfused rabbit lung under normal conditions and during lung injury. Vascular permeability was assessed by measurement of the capillary filtration coefficient (Kf,c). In normal lungs, NTG did not alter Kf,c or the rate of weight gain. Oxidant lung injury was produced by the addition of purine and xanthine oxidase and resulted in increased Kf,c and increased weight gain. However, NTG did not alter these effects of oxidant lung injury. We conclude that NTG does not alter pulmonary vascular permeability in either normal or oxidant-injured lungs.     

Lecithinized superoxide dismutase attenuates phorbol myristate acetate-induced injury in isolated dog lung

Lecithinized superoxide dismutase, a lecithin derivative bound to recombinant human CuZn superoxide dismutase, has a higher affinity for cells such as polymorphonuclear leukocytes and endothelial cells than recombinant human CuZn superoxide dismutase has. We determined the protective effects of lecithinized superoxide dismutase on the increased microvascular permeability induced by phorbol myristate acetate (PMA) in isolated dog lungs. Microvascular permeability was assessed by the capillary filtration coefficient (Kf,c) and solvent drag reflection coefficient (sigma(f)). PMA (13.3 microg) increased microvascular permeability, as evidenced by an increase in Kf,c and the small sigma(f) value. Lecithinized superoxide dismutase at both low (4800 U) and high doses (48,000 U) inhibited the PMA-induced increase in Kf,c, but only the high dose of lecithinized superoxide dismutase attenuated the decrease in sigma(f). Recombinant human CuZn superoxide dismutase did not affect the PMA-induced increase in vascular permeability at either a low (4800 U) or a high dose (48,000 U). These findings suggest that lecithinized superoxide dismutase has a protective effect against oxygen radical-induced lung injury in isolated dog lungs.     

Pattern of injury and the role of neutrophils in reperfusion injury of rat lung

Using a rat lung model, we sought to characterize the time course for ischemia-reperfusion injury and the role of neutrophils in the development of injury. Adult male Long-Evans rats underwent left thoracotomy with dissection and clamping of the left pulmonary artery, bronchus, and vein for 90 min, resulting in complete left lung ischemia. The lungs were then ventilated and reperfused for up to 4 hr. Time-matched sham animals underwent the identical thoracotomy and hilar dissection, but the lungs were not rendered ischemic. Using vascular permeability of 125I-labeled bovine serum albumin as a measure of reperfusion injury, a bimodal pattern of injury was observed. Compared to sham controls, animals undergoing ischemia-reperfusion demonstrated a significant early phase of lung injury at 30 min of reperfusion (P < 0.0001), followed by partial recovery. A second peak of lung injury was noted after 4 hr of reperfusion (P < 0.001). Myeloperoxidase activity in reperfused lung tissue, a measure of neutrophil sequestration, increased during the reperfusion time course. To determine the role of neutrophils in the development of lung reperfusion injury, additional animals undergoing the identical ischemia-reperfusion protocol received either rabbit anti-rat neutrophil serum or preimmune serum the day prior to operation. Profound neutropenia (< 75/mm3 blood) was confirmed by differential leukocyte counts. Neutropenia had no protective effect against microvascular permeability at 30 min of reperfusion, but there was a significant reduction in lung injury at 4 hr (P < 0.005). We conclude that, during lung ischemia-reperfusion, there is a bimodal pattern of injury, consisting of both neutrophil-independent and neutrophil-mediated events.     

In vivo fluorescence microscopy for the assessment of microvascular reperfusion injury in small bowel transplants in rats

With the use of in vivo fluorescence microscopy we have analyzed microvascular reperfusion injury of small bowel isograft transplants in rats. Following 1 hr cold storage in University of Wisconsin solution, the small bowel was transplanted heterotopically, and the intestinal microcirculation was quantitatively analyzed 20-60 min after onset of reperfusion. The intestinal grafts' capillary perfusion of both the mucosa and the circular and longitudinal muscles was not found altered when compared with the intestinal capillary perfusion of sham-operated controls. In contrast, leukocyte-endothelial cell interaction, including leukocyte rolling (40 +/- 5%) and sticking (280 +/- 100 mm-2) in submucosal postcapillary venules, was significantly increased when compared with nontransplanted controls (12 +/- 8% and 20 +/- 10 mm-2, P < 0.01 and P < 0.05, respectively). Leukocyte-endothelial cell interaction was associated with a marked alteration of lymphatic capillary drainage, as indicated by the low functional density of lymphatic microvessels of 10.2 +/- 6.1 cm-1 (P < 0.01 vs. sham-operated controls (39.2 +/- 6.1 cm-1)). From these results we propose that leukocyte-endothelial cell interaction, not capillary "no-reflow," is the primary step in the manifestation of microvascular reperfusion injury following a short period of cold ischemia in small bowel grafts.     

Effect of different solutions of preservatives on the endothelial function of coronary arteries in rats

The effects of cold storage and type of preservation solution on coronary endothelial function are not well established. Experiments were designed to evaluate coronary endothelial-dependent relaxation after a 4-hour cold (4 degrees C) storage in different preservation solutions. Rat hearts, mounted in the Langendorff apparatus, were arrested with a 10-minute perfusion of 4 degrees C crystalloid hyperkaliemic cardioplegic solution (CHCS) (KCl 24 mEq/l) and stored for 4 hours in the following preservation solutions: CHCS (n = 6), Krebs-Ringer solution (KR) (n = 6), 0.9% NaCl (NS) (n = 6) and the University of Wisconsin solution (UW) (n = 6). A fifth group (n = 6) was perfused and stored in UW solution. Endothelium-dependent and independent coronary artery vasorelaxations were respectively tested by infusing 5-hydroxytryptamine (5-HT) (10(-6) mol/l) and sodium nitroprusside (SNP) (10(-5) mol/l) before and after the storage period. In hearts stored with CHCS or KR, coronary artery flow increase to 5-HT and SNP infusions were not significantly affected. However, in hearts preserved with NS or UW solutions, 5-HT coronary response was significantly decreased, indicating endothelial dysfunction. In addition to these findings, coronary flow increase to SNP infusion was decreased in the group perfused and stored with UW, suggesting smooth muscle dysfunction. These experiments suggest that 4-hour cold storage in NS or UW impairs endothelial-dependent coronary relaxation in the isolated rat heart model.     

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.     

Dibutyryl cAMP reduces nonparenchymal cell damage during cold preservation of rat livers

Sinusoidal lining cells are the main target for cold preservation injury and are further damaged with reperfusion. Different agents known to increase intracellular cAMP levels have been shown beneficial. This study was designed to assess the possible protective effect of a cAMP analogue on nonparenchymal cells of rat livers, during cold storage and during reperfusion. Parameters reflecting the status of the liver microvasculature were analyzed. The initial effluent collected after preservation reflects release during the cold storage period; therefore we measured interleukin-1 (IL-1) and endothelin-1 (ET-1) levels in these samples in order to detect and quantitate the degree of activation and/or disruption of Kupffer and sinusoidal endothelial lining cells. Rat livers were harvested after in situ flush with Ringer's lactate with or without 2 mM dibutyryl cAMP, excised, and stored in the same solution at 4 degrees C. After 6 hr, livers were perfused with Krebs-Henseleit buffer for 90 min. Physiological parameters were monitored throughout the perfusion. Perfusate samples were collected every 30 min for RIA measurements of IL-1 and ET-1. Treatment resulted in a significant decrease in release of ET-1 and IL-1 during storage. Likewise, livers treated with cAMP had a significantly improved bile output and decreased portal vein resistance during reperfusion. The beneficial effect granted by the analogue during cold storage and reperfusion was evident on parenchymal and nonparenchymal cells. Levels of ET-1 and IL-1 in the caval effluent confirm and quantitate preservation damage.     

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.     

Ischemic preconditioning enhances donor lung preservation in the rat

BACKGROUND: Ischemic preconditioning achieved by brief periods of ischemia and reperfusion before a prolonged period of ischemia can significantly reduce the extent of cardiac damage in many mammalian species and human beings. In this study we used a rat model of single lung transplantation to show that ischemic preconditioning also occurs in the lung. METHODS: Rats randomly selected for ischemic preconditioning had their left main bronchus and pulmonary artery occluded for 5 minutes, followed by 10 minutes of reperfusion and ventilation. Lungs of control rats were ventilated for 15 minutes. The lungs were perfused with University of Wisconsin solution, then heart and lungs were excised en bloc and stored in University of Wisconsin solution at 0 degree C for 6 or 12 hours. After left pneumonectomy, the left lung of the donor was then implanted into the recipient via left thoracotomy. After 1 hour of ventilation and reperfusion, a right pneumonectomy was performed making the animal completely dependent on the transplanted lung. Samples of arterial blood from the left ventricle were then taken for arterial oxygen tension and arterial carbon dioxide tension determination. Water contents of the donor lungs were measured before and after reperfusion. Thiobarbituric acid reactive substances were measured in the right donor lung after storage. RESULTS: Lungs transplanted after 12 hours of storage had profoundly impaired gas exchange (arterial oxygen tension = 34 +/- 5; arterial carbon dioxide tension = 69 +/- 7 mm Hg) compared with the normal levels in the 6-hour storage group (arterial oxygen tension = 308 +/- 22; arterial carbon dioxide tension = 17 +/- 1 mm Hg). Ischemic preconditioning significantly improved gas exchange in the 12-hour storage group (arterial oxygen tension = 83 +/- 11; arterial carbon dioxide tension = 40 +/- 4 mm Hg). Ischemic preconditioning also significantly decreased thiobarbituric acid reactive substances formation at both 6- and 12-hour storage. CONCLUSIONS: These results show that the phenomenon of ischemic preconditioning occurs in the lung and that it may reduce injury to the donor lung during prolonged cold ischemic storage.     

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.     

Arthroscopic repair of full-thickness tears of the rotator cuff

OBJECTIVE: The authors' goal was to determine the effects of specific binding and blockade of P- and E-selectins by a soluble P-selectin glycoprotein ligand-1 (PSGL-1) in rat models of hepatic in vivo warm ischemia and ex vivo cold ischemia. The authors also sought to determine the effect of selectin blockade on isograft survival in a syngeneic rat orthotopic liver transplant model. SUMMARY BACKGROUND DATA: Ischemia/reperfusion (I/R) injury is a major factor in poor graft function after liver transplantation, which may profoundly influence early graft function and late changes. It is hypothesized that I/R injury leads to the upregulation of P-selectin, which is then rapidly translocated to endothelial cell surfaces within 5 minutes of reperfusion of the liver, initiating steps leading to tethering of polymorphonuclear neutrophil leukocytes to the vascular intima. Local production by leukocytes of interleukin-1, tumor necrosis factor-alpha, or both induces P-selectin expression on the endothelium and continues the cascade of events, which increases cell adherence and infiltration of the organ. METHODS: To examine directly the effects of selectins in a warm hepatic I/R injury model, 100 microg of PSGL-1 or saline was given through the portal vein at the time of total hepatic inflow occlusion. The effects of PSGL-1 in cold ischemia were assessed using an isolated perfused rat liver after 6 hours of 4 degrees C storage in University of Wisconsin (UW) solution, with or without the instillation of PSGL-1 before the storage. To evaluate the effect of selectin blockade on liver transplant survival, syngeneic orthotopic liver transplants were performed between inbred male Sprague-Dawley rats after 24 hours of cold ischemic storage in UW solution. A separate group of animals received two doses of 100 microg of PSGL-1 through the portal vein before storage and before reperfusion of the transplanted liver. Recipient survival was assessed at 7 days, and the Kaplan-Meier product limit estimate method was used for univariate calculations of time-dependent recipient survival events. RESULTS: In an in vivo warm rat liver ischemia model, perfusion with PSGL-1 afforded considerable protection from I/R injury, as demonstrated by decreased transaminase release, reduced histologic hepatocyte damage, and suppressed neutrophil infiltration, versus controls (p < 0.05). When cold stored livers were reperfused, PSGL-1 reduced the degree of hepatocyte transaminase release, reduced neutrophil infiltration, and decreased histologic hepatocyte damage (p < 0.05 vs. UW-only controls). On reperfusion, livers treated with PSGL-1 demonstrated increased portal vein blood flow and bile production (p < 0.05 vs. UW-only controls). In addition, 90% of the rats receiving liver isografts stored in UW solution supplemented with PSGL-1 survived 7 days versus 50% of those whose transplanted syngeneic livers had been stored in UW alone (p < 0.05). CONCLUSIONS: Selectins play an important role in I/R injury of the liver. Early modulation of the interaction between P-selectin and its ligand decreases hepatocyte injury, neutrophil adhesion, and subsequent migration in both warm and cold rat liver ischemia models. In addition, the use of PSGL-1 before ischemic storage and before transplantation prevents hepatic injury, as documented by a significant increase in liver isograft survival. These findings have important clinical ramifications: early inhibition of alloantigen-independent mechanisms during the I/R damage may influence both short- and long-term survival of liver allografts.     

Hysteresis in force probe measurements: a dynamical systems perspective

BACKGROUND: This study was designed to investigate the effects of a modified University of Wisconsin (UW) solution supplemented with one of four buffering agents (histidine, bicine [N,N-bis(2-hydroxyethyl)glycine], tricine [N-tris(hydroxymethyl)methylglycine], and Tris) on liver metabolism during cold ischemic storage. METHODS: Rat livers were flushed and stored for a maximum period of 24 hr at 4 degrees C, and tissue energetics, substrate, and anaerobic end-products were assessed; the group exhibiting the best results during storage was recovered in a 60-min period of warm reperfusion. Relative buffering capacities of the experimental solutions (measured over physiological pH range, in mM H+/L) were: UW, 4.1; histidine+UW, 9.8; Tris+UW, 19.0; bicine+UW, 22.5; tricine+UW, 26.8. RESULTS: In the UW group, ATP levels dropped rapidly over the first 4 hr; 1.0 micromol/g (40% of initial) remained after 4 hr of storage. By 2 hr, ATP levels in bicine- and tricine-treated groups were 0.5 and 1.1 micromol/g greater than in the UW-stored livers and by 10 hr, ATP in bicine-treated livers was twofold that of the control (UW) group. Total adenylate levels also reflected a superior elevation of cellular energetics; even after 24 hr, quantities were 1.4 and 2.0 micromol/g higher than the UW group in bicine- and histidine-supplemented organs. The increase in energetics occurred as a result of increased flux through the major anaerobic energy-producing pathway, glycolysis. The glycolytic rate was significantly greater at storage times > 10 hr with solutions supplemented with bicine, histidine, and tricine. Final values for net lactate accumulation over the entire 24-hr storage period were: UW, 10.1 micromol/g; histidine, 14.3 micromol/g; bicine, 15.2 micromol/g; tricine, 13.8 micromol/g. Activities of glycogen phosphorylase revealed that the activity of this enzyme dropped by 50% within 2 hr of storage in UW. However, histidine and bicine supplementation resulted in a substantial elevation of phosphorylase "a" over 4 hr and 10 hr, respectively. The best buffer of the four examined in this study was bicine; energetics, glycolytic flux, and patterns of adenylate regeneration upon reperfusion were markedly superior to modified UW solution. CONCLUSION: The results of this study suggest that supplementing the "gold standard" UW solution with an additional buffering agent (in order of efficacy: bicine>tricine>histidine) may improve the metabolic status of livers during clinical organ retrieval/storage.     

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.     

Low-dose sodium nitroprusside reduces pulmonary reperfusion injury

BACKGROUND: Reperfusion injury is a significant cause of early allograft dysfunction after lung transplantation. We hypothesized that direct pulmonary arterial infusion of an intravascular nitric oxide donor, sodium nitroprusside (SNP), would ameliorate pulmonary reperfusion injury more effectively than inhaled nitric oxide without causing profound systemic hypotension. METHODS: Using an isolated, ventilated, whole-blood-perfused rabbit lung model, we studied the effects of both inhaled and intravascular nitric oxide during lung reperfusion. Group I (control) lungs (New Zealand White rabbits, 3 to 3.5 kg) were harvested en bloc, flushed with Euro-Collins solution, and then stored inflated for 18 hours at 4 degrees C. Lungs were then reperfused with whole blood and ventilated with 60% oxygen for 30 minutes. Groups II, III, and IV received pulmonary arterial infusions of SNP at 0.2, 1.0, and 5.0 micrograms.kg-1.min-1, respectively, whereas group V was ventilated with 60% oxygen and nitric oxide at 80 ppm during reperfusion. RESULTS: Pulmonary arterial infusions of SNP even at 0.2 microgram.kg-1.min-1 (group II) showed significant improvements in pulmonary artery pressure (31.35 +/- 0.8 versus 40.37 +/- 3.3 mm Hg; p < 0.05) and pulmonary vascular resistance (38,946 +/- 1,269 versus 52,727 +/- 3,421 dynes.s/cm-5; p < 0.05) when compared with control (group I) lungs after 30 minutes of reperfusion. Infusions of SNP at 1.0 microgram.kg-1.min-1 (group III) showed additional significant improvements in dynamic airway compliance (1.98 +/- 0.10 versus 1.46 +/- 0.02 mL/mm Hg; p < 0.05), venous-arterial oxygenation gradient (116.00 +/- 24.4 versus 34.43 +/- 2.5 mm Hg; p < 0.05), and wet-to-dry ratio (6.9 +/- 0.9 versus 9.1 +/- 2.2; p < 0.05) when compared with control (group I) lungs. Lungs that received inhaled nitric oxide at 80 ppm (group V) were significantly more compliant (1.82 +/- 0.13 versus 1.46 +/- 0.02 mL/mm Hg; p < 0.05) than control (group I) lungs. CONCLUSIONS: Pulmonary arterial infusion of low-dose SNP during lung reperfusion significantly improves pulmonary hemodynamics, oxygenation, compliance, and edema formation. These effects were achieved at doses of SNP that did not cause profound systemic hypotension. Direct intravascular infusion of SNP via pulmonary arterial catheters could potentially abate reperfusion injury immediately after allograft implantation.     

Apoptosis after intestinal ischemia-reperfusion injury: a morphological study

BACKGROUND: Apoptosis has been identified after ischemia-reperfusion (IR) injury to the brain, heart, kidney, retina, and the adrenals. Intestinal IR injury causes villous and crypt damage, which has so far been attributed to cellular necrosis. This study was undertaken to investigate the possible role of apoptosis after reperfusion of cold-stored small bowel grafts in syngeneic rats. METHODS: Small intestinal grafts were stored at 4 degrees C for 24 hr in saline (n=6) or in modified University of Wisconsin solution (n=6), followed by reperfusion for 1 hr in syngeneic Lewis rats. Small bowel samples were obtained before storage, after preservation and after 1 hr of reperfusion. They were processed for light and electron microscopy and analyzed for cell death, with particular emphasis on apoptosis. RESULTS: Less than one apoptotic event was seen per 10 crypts in normal and stored bowels. An occasional normal and some denuded villous epithelial cells of stored bowels exhibited apoptosis. After isotransplantation and 1 hr of reperfusion, marked increase in apoptosis was seen in the crypts and denuded villous epithelial cells of both saline- and modified University of Wisconsin-stored bowels. Secondary necrosis was seen in apoptotic cells, as were dark cells. Only a few cells showed signs of primary ischemic necrosis. CONCLUSIONS: Apoptosis occurs after intestinal IR injury. Modulation of its genetic regulatory and biochemical effector machinery might alleviate or even prevent IR injury in small bowel transplanted after similar periods of storage.     

Tumor necrosis factor-alpha in ischemia and reperfusion injury in rat lungs

The effects of both recombinant rat tumor necrosis factor-alpha (TNF-alpha) and an anti-TNF-alpha antibody were studied in isolated buffer-perfused rat lungs subjected to either 45 min of nonventilated [ischemia-reperfusion (I/R)] or air-ventilated (V/R) ischemia followed by 90 min of reperfusion and ventilation. In the I/R group, the vascular permeability, as measured by the filtration coefficient (Kfc), increased three- and fivefold above baseline after 30 and 90 min of reperfusion, respectively (P < 0.001). Over the same time intervals, the Kfc for the V/R group increased five- and tenfold above baseline values, respectively (P < 0.001). TNF-alpha measured in the perfusates of both ischemic models significantly increased after 30 min of reperfusion. Recombinant rat TNF-alpha (50,000 U), placed into perfusate after baseline measurements, produced no measurable change in microvascular permeability in control lungs perfused over the same time period (135 min), but I/R injury was significantly enhanced in the presence of TNF-alpha. An anti-TNF-alpha antibody (10 mg/rat) injected intraperitoneally into rats 2 h before the lung was isolated prevented the microvascular damage in lungs exposed to both I/R and V/R (P < 0.001). These results indicate that TNF-alpha is an essential component at the cascade of events that cause lung endothelial injury in short-term I/R and V/R models of lung ischemia.