Euro-Collins Solution Research Articles

Mitochondrial Injuries in Rat Lungs Preserved for 17 h: An Ultrastructural Study

Mitochondria of the small vasculature endothelial cells were examined in preserved rat lungs before and after reperfusion, and the ultrastructural changes were correlated with pulmonary function after reperfusion. Rat lungs were flushed with perfusate and prostaglandin E1 and divided into five groups (n = 5 in each group): group A, normal control group; group B, University of Wisconsin solution; group C, Euro-Collins solution; group D, ET-Kyoto solution, and group E, new ET-Kyoto solution. After preservation at 4°C for 17 h, the left lungs were reperfused at 37°C for 60 min. Tissue was sampled and mitochondria of the small vasculature endothelial cells were ultrastructurally analyzed by transmission electron microscopy before and after reperfusion. The ultrastructure of the mitochondria was well maintained in groups A, B and E before and after reperfusion. In group C, the number of severely degenerated mitochondria in the sectional area of 100 μm² before reperfusion was 18.0 ± 3.9, which was significantly larger than in the other groups (p < 0.01), and the total number of mitochondria significantly decreased with reperfusion (from 24.8 ± 3.5 to 8.2 ± 2.4, p < 0.05). In group C, the shunt fraction, mean pulmonary arterial pressure and the wet-dry ratio of the lung tissue after reperfusion C were significantly higher than in the other groups (p < 0.05; 76.3 ± 1.5%, 54.8 ± 4.2 mm Hg, and 20.6 ± 2.5, respectively). A positive correlation was found between the percentage of the mitochondrial degeneration before reperfusion and the physiological parameters after reperfusion. Mitochondrial damage associated with cold ischemia is probably involved in lung injury caused by cold preservation and reperfusion.

Histological Evaluation of Proximal Tubule Cell Injury in Isolated Perfused Pig Kidneys Exposed to Cold Ischemia

Background.Ischemic injury of the renal allograft before transplantation is a major cause of impaired graft function. Proton nuclear spectroscopy provides a useful technique for evaluating proximal tubular activity. In addition to this technique, we proposed a histological grading system for quantifying proximal tubule alterations.Methods.The aim of this study was to evaluate the histological lesions of tubule epithelial cells in the model of isolated perfused pig kidneys following 48 to 72 h of cold storage in Euro-Collins solution. Normothermic isolated perfused pig kidneys were randomized in three experimental groups : Group 1, control group; cold flush with cold heparinized solution followed by immediate reperfusion (n= 6); Group 2, 48 h of cold storage in Euro-Collins followed by reperfusion (n= 6); Group 3, 72 h of cold storage in Euro-Collins followed by reperfusion (n= 6). Proton nuclear spectroscopy of urine and biochemical studies were performed for whole renal functional evaluation during reperfusion. Optical and electron microscopy analyses of the reperfused kidneys were performed by four investigators and the degree of cell injury was assessed using 8 different criteria in a 5-scale numerical score.Results.Numerical scores corroborate the results from NMR spectroscopy and differed significantly between the three groups studied. The degree of proximal tubule cell damage was increased with prolonged cold ischemia as shown particularly in Group 3.Conclusion.The results from this study showed that analysis of cell injury based on an histological grading system in the model of isolated perfused kidney allows the quantification of the degree of proximal tubule injury. Thus, such morphological system analysis could be a useful method for quantifing tubule cell injuries observed under various physiopathological conditions.

Improvement of Rat Lung Structure and Function after Preservation with Celsior

Ischemia/reperfusion-induced increase in pulmonary microvascular permeability was shown to be reduced after preservation with Celsior. We investigated reimplantation-induced lung injury in isolated, reperfused rat lungs after preservation via the pulmonary artery with Celsior, Celsior + prostacyclin, and reduced-potassium (40 mmol) Euro-Collins solution (40 ml/kg/body wt each) followed by 2 h of cold ischemia. Arterial and veneous oxygen tensions were recorded during 50 min ofin vitroreperfusion after which the lungs (10 right lungs per experimental group) were fixed by vascular perfusion. The tissue was further processed for microscopy, and histological changes were quantified stereologically. Lung preservation with Celsior resulted in a significantly higher volume of air-filled alveolar space with a large proportion of widely distended alveoli compared with the other groups. In the Euro-Collins group the fraction of atelectatic alveoli exceeded that observed in Celsior-preserved lungs. In accordance, the difference between arterial and venous oxygen tensions was significant among Euro-Collins- and Celsior-protected lungs, with improved oxgenation values in the Celsior group. In contrast, addition of prostacyclin to Celsior treatment resulted in rather variable structural as well as functional data. There were no differences in the volumes of intraalveolar edema among the groups tested. However, the volume of alveolar tissue was increased in the Euro-Collins group. In conclusion, compared with Euro-Collins and Celsior + prostacyclin solutions, preservation with Celsior resulted in improved structural characteristics which in combination with improved oxygenation parameters supports the prospective advantage of Celsior in clinical organ preservation.

Influence of different routes of flush perfusion on the distribution of lung preservation solutions in parenchyma and airways.

The present study was performed to investigate the influence of different routes of perfusion on the distribution of the preservation solutions in the lung parenchyma and upper airways. Pigs were divided into four groups: control (n = 6), pulmonary artery (PA) (n = 6), simultaneous PA + bronchial artery (BA) (n = 8), and retrograde delivery (n = 6). After preparation and cannulation, cardioplegia solution and Euro-Collins solution (ECS) for lung preservation were given simultaneously. After removal of the heart, the double lung bloc was harvested. Following parameters were assessed: total and regional perfusion (dye-labeled microspheres), tissue water content, PA, aorta, left atrial and left ventricular pressures, cardiac output and lung temperature. Our data show that flow of the ECS in lung parenchyma did not reach control values (9.4+/-1.0 ml/min per g lung wet weight) regardless of the route of delivery (PA 6.3+/-1.5, PA + BA 4.8+/-0.9, retrograde 2.7+/-0.9 ml/min per g lung wet weight). However, flow in the proximal and distal trachea were significantly increased by PA + BA delivery (0.970+/-0.4, respectively, 0.380+/-0.2 ml/min per g) in comparison with PA (0.023+/-0.007, respectively, 0.024+/-0.070 ml/min per g), retrograde (0.009+/-0.003, respectively, 0.021+/-0.006 ml/min per g) and control experiments (0.125+/-0.0018, respectively, 0.105+/-0.012 ml/g per min). Similarly the highest flow rates in the right main bronchus were achieved by PA + BA delivery (1.04+/-0.4 ml/min per g) in comparison with 0.11+/-0.03 in control, 0.033+/-0.008 in PA, and 0.019+/-0.005 ml/min per g in retrograde group. Flows in the left main bronchus were 0.09+/-0.02 ml/min per g in control, 0.045+/-0.012 ml/min per g in PA, and 0.027+/-0.006 ml/min per g in retrograde group. The flow rates were significantly (P = 0.001) increased by PA + BA delivery of the storage solution (0.97+/-0.3 ml/min per g). Our data show that the distribution of ECS for lung preservation is significantly improved in airway tissues (trachea and bronchi) if a simultaneous PA + BA delivery is used.

Perfusão pulmonar anterógrada "versus" retrógrada na preservação pulmonar para transplante em modelo canino de viabilidade pulmonar pós-morte

A doação pulmonar após parada cardiocirculatória tem sido estudada experimentalmente na obtenção de órgãos para transplante, porém a severa lesão isquêmica/reperfusão exigem métodos de preservação que permitam viabilidade pulmonar. A perfusão do enxerto com solução cristalóide hipotérmica via anterógrada (artéria pulmonar) é o método de preservação mais utilizado, porém esta via não perfunde a circulação brônquica, permitindo a retenção sanguínea neste território capaz de desencadear fenômenos de lesão de reperfusão. Isto nos levou a testar os efeitos da perfusão anterógrada versus retrógrada (via átrio esquerdo, capaz de perfundir a circulação brônquica) em modelo canino de transplante unilateral cujos pulmões foram extraídos 3 horas após parada cardiorrespiratória. Doze cães doadores foram sacrificados com tiopental sódico e mantidos à temperatura ambiente sob ventilação mecânica durante 3 horas, após as quais os animais foram randomizados e os blocos cardiopulmonares perfundidos via retrógrada (n = 6) ou anterógrada (n = 6) com solução de Euro-Collins modificada e extraídos. Os receptores (n = 12) foram anestesiados, pneumonectomizados e submetidos a transplante pulmonar esquerdo recebendo enxertos perfundidos por via retrógrada (grupo I) ou anterógrada (grupo II). Após a reperfusão do enxerto, os animais foram mantidos sob ventilação mecânica (FiO2 = 1) por 6 horas, sendo então sacrificados. Durante este período obtiveram-se medidas hemodinâmicas e gasometrias arteriais, além de amostras de tecido pulmonar para dosagem de ATP intracelular. As medidas hemodinâmicas não diferiram entre os grupos. Nos animais do grupo I a PaO2 e PaCO2 foram superiores às do grupo II (p = 0,016 e p = 0,008, respectivamente). O ATP intracelular não diferiu entre os grupos, embora tenha se reduzido nas amostras obtidas na extração do enxerto do doador quando comparados aos valores após a reperfusão (p = 0,01) e ao término do período de avaliação (p = 0,01). Os autores concluem que, neste modelo experimental, a perfusão retrógrada hipotérmica resulta em função superior do enxerto após 3 horas de isquemia normotérmica sob ventilação mecânica.

Cl-Esterase-inhibitor ameliorates reperfusion injury of the lung

334 This study was designed to determine a possible beneficial effect on reperfusion injury of the lung by Cl-Esterase Inhibitor ClINH. Methods: In 12 Minipigs the left lung was selectively perfused with cold Euro-Collins solution. After 90 minutes of warm ischemia the lungs were reperfused and the contralateral pulmonary artery and main bronchus clamped. 6 pigs were used as controls. In the ClINH group (6 pigs) 150 units/kg ClINH concentrate were administered 30 minutes prior to reperfusion. Hemodynamic and respiratory parameters were monitored for 7 hours. Surfactant analysis of bronchoalveolar lavage was performed prior to preservation and 2 hours after reperfusion. Results: In the control group reperfusion injury developed in all cases. Lung function deteriorated with decreasing arterial PO2, reduction of compliance (C) and increase of pulmonary vascular resistance (PVR). Right heart failure developed within 234 ± 83 minutes. In the Cl-INH group increase of PVR was less (p=0.027). C (p=0.016) as well as arterial PO2 were significantly higher (p=0.047). All but one pig survived 7 hours of reperfusion (p=0.009). Cl-INH activity dropped in the control group from 62.5±8% preOP to 51±8% after 2 hours of reperfusion. In the ClINH group levels were 63.8±6%, and 176±8% respectively. Surfactant function and composition was impaired in both groups. Conclusion: During reperfusion injury a reduction of ClINH activity was found in controls. Substitution of ClINH reduces significantly reperfusion injury of the lung but did not improve surfactant function.

Expression of Stress Proteins, Adhesion Molecules, and Interleukin-8 in Endothelial Cells after Preservation and Reoxygenation

Endothelial activation is a central feature of preservation-induced allograft injury. The present study aims at a quantitative assessment of stress proteins, adhesion molecules, and interleukin-8 in a cell culture-based model of organ preservation. Human umbilical vein endothelial cells were exposed to cold, hypoxic storage in University of Wisconsin (UW), histidine-tryptophane-ketoglutarate (HTK), and EuroCollins solutions for 8 h with subsequent rewarming/reoxygenation (rew/reox) for 1 and 4 h. A cell-based ELISA was designed for detection of heat shock proteins (HSP) 60 and 70, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (ELAM-1). Immunohistochemical staining was performed for comparison. Interleukin-8 was quantified by ELISA. HSP 70 was expressed after cold storage in HTK and EuroCollins solution and after rew/reox in all groups. A constitutive expression of HSP 60 was observed with further upregulation after rew/reox following cold storage in all experimental groups. ICAM-1 was clearly upregulated, but VCAM-1 showed only weak expression after cold storage and rew/reox. ELAM-1 was detectable in minimal amounts after cold storage but was considerably upregulated after 4 h of rew/reox. A significant increase of interleukin-8 release could be found after 4 h of rew/reox following storage in EuroCollins solution. Expression of stress proteins can be considered as a new parameter of preservation-associated endothelial activation. Apart from possible protective effects, allograft vasculopathy could be in part a consequence of the antigeneic potential of heat shock proteins connected with effects caused by adhesion molecules and inflammatory cytokines.

Modulation of reperfusion injury after single lung transplantation by pentoxifylline, inositol polyanions, and sin-1

Objective: Previous studies have suggested reductions in lung reperfusion injury with pentoxifylline, inositol polyanions, and the nitric oxide donor, SIN-1, but these agents have never been directly compared to ascertain which is superior. We investigated these agents in a porcine model of left single lung transplantation. Methods: Donor lungs were preserved with modified Euro-Collins solution for a mean ischemic time of 18.4 hours. Neutrophil trapping in the graft, pulmonary vascular resistance, free radical release (measured by malonaldehyde levels) and gas exchange were assessed over a 12-hour period. All groups were reperfused at an initial pulmonary artery pressure of 20 mm Hg. Group A (n = 5) was a control group with no interventions added; group B was reperfused with the addition of intravenous inositol polyanions (0.02 mg/kg/h), and group C was reperfused with intravenous SIN-1 (0.02 mg/kg/h). Group D was reperfused with the addition of intravenous pentoxifylline (2 mg/kg/h). Results: Neutrophil sequestration was observed within 10 minutes of reperfusion in group A. This was attenuated significantly by interventions in groups B, C, and D. In group D, malonaldehyde levels were significantly lower than in other groups and was associated with superior oxygenation. Pulmonary vascular resistance was reduced in groups B, C, and D compared with group A. Conclusions: Pentoxifylline, when administered only to recipient animals was superior to the other interventions studied. Inositol polyanions are promising as a possible therapeutic intervention but were not as effective as the other agents studied. (J Thorac Cardiovasc Surg 1999;117:556-64)

Does PGE 1 attenuate potassium-induced vasoconstriction in initial pulmonary artery flush on lung preservation?

The standard clinical protocol for lung transplantation employs cold single pulmonary artery flush with Euro-Collins solution or the University of Wisconsin solution. Prostaglandin E 1 (PGE 1) is usually given by direct injection into the pulmonary artery to reduce pulmonary vasoconstriction caused by these intracellular, high-potassium solutions, however, the efficacy of PGE 1 on lung preservation remains controversial. In this study we demonstrated that vasodilator effects of PGE 1 were markedly reduced under a high-potassium condition, and that potassium-induced pulmonary vasoconstriction were inhibited by calcium channel blocker nifedipine. There are three therapeutic options in the cold single pulmonary artery flush for optimal lung transplantation, including the use of a higher dose of PGE 1, use of the calcium channel blocker instead of PGE 1, or the use of the extracellular, low-potassium solution such as low-potassium dextran solution for initial pulmonary artery flush before the lung harvest.

Effect of various short-term storage methods on viability of cancellous bone fragments

Abstract Objective To determine effects of various storage methods on ex vivo viability of cancellous bone fragments. Sample Population Cancellous bone fragments obtained from 4 New Zealand White rabbits. Procedure Cancellous bone fragments were stored for 3 hours on ice in 1 of 5 preservation solutions or in 0.9% NaCI. Fragments were then reperfused (37 C) in oxygenated physiologic buffer solution for 1 hour. Cellular viability in fragments was assessed by ethidium monoazide labeling and fluorescence microscopy. Results Viability in fresh cancellous bone ranged from 85 to 100% (mean ± SEM, 88 ± 7%). Storage of fragments significantly reduced viability. Viability in bone fragments stored at 22 C in blood-soaked sponges or 0.9% NaCI solution was 63.8 ± 3 and 65.2 ± 7%, respectively. Use of cold 0.9% NaCI solution reduced viability to 53.6 ± 3%. Viability was significantly better for fragments stored in cold phosphate-buffered sucrose (70.4 ± 2%) and EuroCollins (71.4 ± 3%) solutions. After warm reperfusion, viability was best for fragments stored in cold phosphate-buffered sucrose (70.2 ± 4%), EuroCollins (72.6 ± 3%), or UW lactobionate solutions (69.6 ± 3%), compared with those stored in cold 0.9% NaCI (47.6 ± 3%) or hypertonic citrate (53.0 ± 3%) solutions or blood-soaked sponges (57.2 ± 3%). Conclusions Hypothermic storage in solutions designed to prevent temperature-dependent cell injury were best for maintaining cancellous bone fragment viability. Clinical Relevance Hypothermia may be advantageous for use in storing cancellous bone fragments during procedures that dictate a prolonged period between harvest and placement of graft fragments. (Am J Vet Res 1999;60:63–67)

Effect of coenzyme Q10 on superoxide production in rats with reperfusion injuries.

We examined the effect of coenzyme Q10 (Co Q10) on superoxide radical (O2-) production in a model of rat reperfusion injury. The chemiluminescence method using a derivative of luciferin was used to quantify O2- production by erythrocytes in the reperfused limb after a period of ischaemia. A total of 20 limbs from Lewis rats were preserved at 4 degrees C in Euro-Collins solution for 72 hours, and were grafted orthotopically to syngeneic rats by a microsurgical technique. In the treated group (n = 10), Co Q10 (10 mg/kg) was injected intraperitoneally into the recipients one hour before reperfusion. In the control group (n = 10), the same dose of solvent was given. To measure the extent of oxidative stress, heparinised blood from the treated and control recipients was collected before, and at 15, 30, and 60 minutes after reperfusion for the measurement of chemiluminescence. O2- production in the Co Q10-treated group was significantly lower than in the control group (p < 0.05). Although these findings suggest that Co Q10 scavenged O2- that was produced in the replanted limbs as a result of ischaemia-reperfusion injury, we should consider other possible mechanisms by which this agent may protect against ischaemia-induced reperfusion injury.

Hypothermic preservation of isolated rat lungs in modified bicarbonate buffer, EuroCollins solution or St Thomas' Hospital cardioplegic solution.

Inadequate preservation solutions limit lung storage times and, consequently, transplant programs. To address this problem we established an isolated, ventilated and perfused rat lung preparation. Here we report the effects of hypothermic storage in EuroCollins solution, St Thomas' Hospital cardioplegic solution and a modified bicarbonate buffer solution. Lungs from male Wistar rats (230-330 g) were perfused via the pulmonary artery with modified bicarbonate buffer (37 degrees C, 15 ml/min, constant flow) and ventilated by positive pressure (tidal volume:1.6-1.8 ml, 80 breaths/min). Vascular resistance (pulmonary artery pressure:perfusate flow ratio) and airways compliance (tidal volume:tracheal pressure ratio) were measured. After a control perfusion period (20 min), lungs were flushed with, then immersed in, bicarbonate buffer (4 degrees C) for varying periods (0-24 h). After storage, lung function was assessed during 20 min reperfusion. Having established a suitable period for study, storage in EuroCollins, St Thomas' Hospital cardioplegic solution or bicarbonate buffer were compared. Pulmonary compliance (ml/cmH2O) was significantly (P < 0.05) reduced in lungs stored for 6 h in modified bicarbonate buffer (0.026 +/- 0.008), EuroCollins solution (0.013 +/- 0.002) or St Thomas' Hospital solution (0.025 +/- 0.005) compared to unstored lungs (0.068 +/- 0.007). Vascular resistance, (1.32 +/- 0.13 cmH2O/ml per min) in unstored lungs, was similar in lungs stored in St Thomas' Hospital solution but increased significantly in lungs stored in modified bicarbonate buffer (3.22 +/- 0.78 cmH2O/ml per min) or EuroCollins solution (4.66 +/- 0.57 cmH2O/ml per min). Hypothermic storage of rat lungs for 6 h in modified bicarbonate buffer or St Thomas' Hospital solution causes less increase in vascular resistance on reperfusion than EuroCollins solution.

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 E 1 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°C for 18 hours before reperfusion. Groups 3 and 4 lungs were flushed with EC solution containing 200 μmol/L 8-bromo-cGMP and stored at 4°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 (E 1) 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.

A stable prostacyclin analog, beraprost sodium, attenuates platelet accumulation and preservation-reperfusion injury of isografts in a rat model of lung transplantation.

Recent studies have shown that the extent of platelet accumulation in the vasculature of transplanted organs correlates with the degree of preservation-reperfusion injury. In this study, we examined the effect of a stable prostacyclin analog, beraprost sodium, which possesses potent antiplatelet activity, on parameters of platelet accumulation and preservation-reperfusion injury of isografts in a rat model of lung transplantation. The heart-lung blocks of donor rats were flushed with and preserved in modified Euro-Collins solution at 4 degrees C for 6 hr or 24 hr. The left lung was transplanted into recipient rats and reperfused for 1 hr. Lung injury was evaluated by the pulmonary blood flow ratios to the lung isografts, the weight gain of the isografts, and histological examination. Small portions of the lung isografts were excised and stained with an antibody specific for rat platelets. A scoring system was developed to semiquantitate the intensity of antibody staining (score 0-4). The recipient rats received oral administration of beraprost sodium (0.3 mg/kg) before lung transplantation. Control animals received no beraprost sodium. In the 6-hr preservation study, administration of beraprost sodium significantly reduced the score for platelet accumulation (1.8+/-0.4 vs. 3.3+/-0.5, P<0.01). This observation was accompanied by a significantly decreased degree of preservation-reperfusion injury as evidenced by an increased blood flow ratio (13.7+/-2.6% vs. 4.5+/-3.6%, P<0.01) and a reduced weight gain (0.7+/-0.2 g vs. 1.1+/-0.2 g, P<0.01). Histological examination revealed severe capillary congestion in three of six cases in the control group, while no capillary congestion was observed in the beraprost group. In the 24-hr preservation study, no differences were seen in platelet accumulation scores or parameters of lung injury. Beraprost sodium, an antiplatelet agent, reduces platelet accumulation and preservation-reperfusion injury of lung transplants at 6 hr in this rat isograft model.

PROTECTIVE EFFECT OF ISCHEMIC-PRECONDITIONING ON LIVER PRESERVATION-REPERFUSION INJURY IN RATS

244 The current study was designed to evaluate the potential ischemic-preconditioning (IP) of the liver graft to protect the liver graft from subsequent long-term preservation-reperfusion injury. Methods: IP was done by a temporary interruption of the portal vein and hepatic artery followed by reflow of hepatic circulation. Liver were transplanted after preservation of the liver graft for 16hr, 20hr, or 24 hr. NAME, L-arginine, or adenosine was administered to block or stimulate synthesis of nitric oxide (NO) in the rats of long-term preserved liver grafts. Serum rat TNF and liver graft apoptosis were determined by bioassay and TUNEL method respectively. Results: After a 16 hr cold ischemia time of the liver grafts in Euro-Collins solution, two of ten rats (20%) survived for over one day. However, 75% of ischemic-preconditioning liver grafts (n=8) survived for over 5 days with a improvement of liver function compared to the control animals (10%, five day survival, n=10). Four of six recipients of 20hr preserved liver grafts survived for over five days. Blockage of NO pathway using NAME, attenuated the protective effect of ischemic-preconditioning on the liver preservation-reperfusion injury. Administration of either L-arginine or adenosine (NO precursor) would prolonged the survival of 16hr-preserved liver grafts. Short-term ischemic-preconditioning (5-10 group) slightly increased survival of 16hr-preserved liver grafts while four of five rats with 16hr preserved liver grafts survived for over 5 days when short-term IP plus L-arginine. However, prolonged ischemic-preconditioning (20-10 group) resulted in a damage of liver function. The levels of serum TNF were significantly decreased in IP treated group compared to untreated rats of long-term preserved liver grafts. Higher numbers of apoptotic cells were observed in the untreated animals(226.25±100.65cells/10mm2)but not in IP treated rats of long-term preserved liver grafts (20.8±22.6cells/10mm2) (P<.05). Conclusion. The current results shows that ischemic-preconditioning protects the liver graft from subsequent long-term liver preservation-reperfusion injury in a rat liver transplantation model. The protective role of ischemic preconditioning may be mediated by endogenous production of NO that inhibits TNF induced hepatocyte apoptosis.

Comparison of Euro-Collins Solution, Low-Potassium Dextran Solution Containing Glucose, and ET-Kyoto Solution for Lung Preservation in an Extracorporeal Rat Lung Perfusion Model

We have recently developed a modified ET-Kyoto solution by adding N-acetylcysteine, nitroglycerin, and dibutyryl cyclic adenosine monophosphate to the previously reported ET-Kyoto solution. The purpose of this study was to compare the new ET-Kyoto solution with the regular Euro-Collins solution and a low-potassium dextran solution containing glucose (Perfadex^®) in an isolated rat lung perfusion model. Rat lung blocks were preserved with ET-Kyoto solution, with Euro-Collins solution, or with Perfadex for 4 h. Arterial oxygen tension, shunt fraction, peak inspiratory pressure, mean pulmonary arterial pressure, and pulmonary vascular resistance were assessed up to 50 min after reperfusion. ET-Kyoto solution provided a significantly better lung function after reperfusion than Euro-Collins solution and Perfadex, while Perfadex was also superior to the Euro-Collins solution. We conclude that lung preservation with ET-Kyoto solution is significantly superior to Euro-Collins solution and to Perfadex in an isolated rat lung perfusion model.

Controlled reperfusion prevents pulmonary injury after 24 hours of lung preservation

Background. Posttransplantation lung reperfusion injury continues to be a major problem. We have shown that controlling the initial period of reperfusion limits this injury after 2 hours of warm lung ischemia. The effectiveness of this modality, however, is unknown after longer periods of cold ischemia, which more closely mimics the clinical situation. Methods. After baseline measurements, 10 pigs had the left lung flushed with a modified Euro-Collins solution, explanted, stored at 4°C for 24 hours, and transplanted into 10 other pigs. Five (group 1) underwent uncontrolled reperfusion created by removal of the vascular clamps after implantation of the new left lung, mimicking the clinical situation. The other five (group 2) underwent controlled reperfusion, which we performed by taking blood from the femoral artery, mixing it with a crystalloid solution (using a mixer heater) to make the blood hyperosmolar, alkalotic, and substrate-enriched, and pumping it through a leukocyte-depleting filter into the transplanted lung for 10 minutes at a pressure of 20 to 30 mm Hg before removing the pulmonary artery clamp. The right pulmonary artery and bronchus were then ligated, and left lung function was assessed each hour for 4 hours and compared with baseline. Results. Controlled reperfusion (group 2) minimized the reperfusion injury, preserving posttransplant pulmonary compliance (92% ± 1% versus 68% ± 1%; p < 0.001), reducing the rise in pulmonary vascular resistance (27% ± 2% versus 166% ± 3%; p < 0.001), improving oxygenation (P o 2, 425 ± 14 versus 82 ± 11 mm Hg; p < 0.001), and lowering myeloperoxidase activity (0.22 ± 0.02 versus 0.45 ± 0.02 ΔOD/mg protein per minute; p < 0.001) and tissue edema (83.0% ± 0.3% versus 84.9% ± 0.3%; p < 0.001) compared with uncontrolled reperfusion, which resulted in an injury so severe that 3 of 5 pigs died before the 4-hour measurements. Conclusions. After 24 hours of cold ischemia uncontrolled reperfusion results in a severe pulmonary reperfusion injury. This injury is almost completely avoided by controlling the composition (modified solution and white blood cell filter) and conditions (pressure) of the reperfusion. Because this experiment mimics the clinical situation, it suggests surgeons should begin to use this modality to limit reperfusion injury after lung transplantation.

Evaluation of 18-hour lung preservation with oxygenated blood for optimal oxygen delivery

Background. Previous studies have shown that availability of oxygen during lung preservation to maintain aerobic metabolism may be essential for the optimal viability of preserved lung tissue. The purpose of this study was to evaluate lung preservation with oxygenated blood for optimal oxygen delivery to the lung graft in a rabbit model. Methods. Eighteen excised rabbit lungs were flushed and stored for 18 hours at 10°C with one of the following: Euro-Collins solution (EC; n = 6), oxygenated homologous blood (OB; n = 6), or low-potassium dextran solution (LPD; n = 6). Poststorage lung functions were evaluated with isolated, blood-perfused lung model for 10 minutes. Results. The mean oxygen tensions after reperfusion for the EC, OB, and LPD groups (47.0 ± 2.8, 76.9 ± 13.1, 96.2 ± 10.9 mm Hg at 10 minutes, respectively) were significantly different throughout the perfusion period (EC < OB < LPD, p < 0.05; EC < LPD, p < 0.01). Pulmonary artery pressure during the reperfusion period in the EC group (35.8 ± 4.4 mm Hg at 10 minutes) was higher than that in the OB and LPD groups (29.8 ± 4.3 and 22.4 ± 2.2 mm Hg, respectively) (EC > OB, EC > LPD, p < 0.05). However, the E-selectin level in the reperfused blood in the OB group (5.04 ± 0.24 ng/mL) was significantly elevated compared with that in other groups (EC, 3.56 ± 0.54; LPD, 2.92 ± 0.35 ng/mL, p < 0.05), which indicated enhanced neutrophil recruitment in the OB group. Comparisons of thrombomodulin and endothelin among the three groups did not reach statistical significance. Conclusions. We conclude that OB may enhance lung preservation as compared with EC solution, probably through its enriched oxygen delivery during storage and extracellular composition. However, the availability of oxygenated blood does not exceed that of LPD solution because of augmented neutrophil recruitment, which may activate neutrophil–endothelial interactions.

Protective effect of ischemic preconditioning on liver preservation-reperfusion injury in rats.

Ischemic-preconditioning is a process whereby a brief ischemic episode confers a state of protection against subsequent long-term ischemia-reperfusion injury. Ischemic preconditioning has been studied in heart and liver ischemia-reperfusion injury; however, few studies have been performed in the model of preservation-reperfusion injury in liver transplantation. The current study was designed to evaluate the ability of ischemic preconditioning to protect liver grafts from long-term preservation-reperfusion injury. Male Sprague Dawley rats were used as donors and recipients of orthotopic liver transplantation. Ischemic preconditioning was done by interruption of the portal vein and hepatic artery for 5, 10, and 20 min (5-10, 10-10, and 20-10 groups). Reflow was initiated by removal of the clamp for another 10 min in all groups. The liver was removed and placed in a bath with Euro-Collins solution for different preservation times. Tolerance of the transplanted liver to cold ischemia was determined by survival time and liver function tests. Rat tumor necrosis factor was analyzed by a bioassay. Nomega-Nitro-L-arginine methyl ester, L-arginine, or adenosine was administered to block or stimulate the synthesis of nitric oxide (NO) in the rats that received long-term-preserved liver grafts. Twenty percent of syngeneic rats (n=10) that received a liver graft with a 16-hr cold ischemia time in Euro-Collins solution survived for more than 1 day and 10% survived for more than 5 days. In contrast, 87.5% of rats (n=8) that received a liver graft with ischemic preconditioning (10-10 group) and 16 hr of cold ischemia survived for more than 1 day and 75% for more than 5 days. Recipients of liver grafts with ischemic preconditioning had significantly reduced levels of serum aspartate transaminase and tumor necrosis factor-alpha, as well as increased bile flow, compared with recipients of liver grafts without ischemic preconditioning. Blockage of the NO pathway using Nomega-nitro-L-arginine methyl ester, a stereospecific competitive inhibitor of NO formation, attenuated the protective effect of ischemic preconditioning. Administration of one of two precursors of NO synthesis, L-arginine or adenosine, prolonged the survival of rats that received 16-hr-preserved liver grafts. In addition, L-arginine synergized with short-term ischemic pre conditioning (5-10 group) to increase the survival of rats that received a liver graft with a 16-hr cold ischemia time, and the survival rate was 83% after 5 days. Finally, prolonged ischemic preconditioning (> or = 20 min; 20-10 group) resulted in liver damage and loss of function. The current results show that ischemic preconditioning protects the liver graft from subsequent long-term cold preservation-reperfusion injury in a rat liver transplantation model. The protective role of ischemic preconditioning may be mediated by the endogenous production of NO.

Addition of aprotinin to organ preservation solutions decreases lung reperfusion injury

Background. Organ preservation injury is associated with endothelial cell damage, destabilization of mitochondrial and cell membranes, and the release of proteolytic enzymes. In addition to its well-known clinical effect of reducing perioperative blood loss, aprotinin has antiproteolytic and membrane-stabilizing properties. We hypothesized that adding aprotinin to Euro-Collins (EC) and University of Wisconsin (UW) solutions would decrease preservation injury in cultured endothelial cells and a whole organ rat lung model. Methods. Bovine aortic endothelial cells were cultured and stored in the respective solution at 4°C for 12 or 48 hours. Endothelial cell viability after storage was assessed by dimethylthiazole tetrazolium cytotoxicity assay. In the whole organ model, rat lungs were isolated, flushed with the respective solution, and stored at 4°C for 6 or 12 hours. The lungs were ventilated with 100% O 2 and reperfused with fresh blood. Alveolar–arterial O 2 difference, O 2 tension, capillary filtration coefficient, and compliance were determined. Results. Endothelial cell viability was optimized with the addition of aprotinin to EC and UW at a dose of 150 KIU/mL (0.02 mg/mL). In the isolated perfused lung model, after 6 hours of ischemic storage, aprotinin-enhanced (100 KIU/mL [0.014 mg/mL]) EC and UW decreased alveolar–arterial O 2 difference, increased O 2 tension, and decreased capillary filtration coefficient compared with EC and UW alone. After 12 hours of ischemic storage, aprotinin-enhanced EC and UW decreased alveolar–arterial O 2 difference, increased O 2 tension, decreased capillary filtration coefficient, and increased compliance compared with EC and UW alone. Conclusions. The addition of aprotinin to EC and UW solutions increases endothelial cell viability in hypoxic cold storage conditions. In terms of whole organ function, aprotinin improves lung preservation as demonstrated by increased oxygenation and compliance, and decreased capillary permeability. This study is clinically applicable as there is already extensive experience with the use of aprotinin in heart and lung transplant recipients, in addition to its routine use in conventional cardiac operations.