Ann Thorac Surg 1997;64:814-820
© 1997 The Society of Thoracic Surgeons
Original Article: General Thoracic
Donor Treatment With the Lazeroid U74389G Reduces Ischemia–Reperfusion Injury in a Rat Lung Transplant Model
Bernard Hausen, MD,
Peter Mueller,
Marcus Bahra,
Raj Ramsamooj, MD,
Randall E. Morris, MD,
Charles W. Hewitt, PhD
Division of Thoracic and Cardiovascular Surgery, Surgical Center, Hannover Medical School, Hannover, Germany; Division of Surgical Research, Department of Surgery, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School at Camden, Camden, New Jersey; and Transplantation Immunology, Department of Cardiothoracic Surgery, Stanford University Medical Center, Stanford, California
Accepted for publication March 13, 1997.
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Abstract
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Background. Antioxidant treatment with lazeroids has proven beneficial for the amelioration of reperfusion injury in experimental lung transplantation. This study compares the effect of donor versus recipient treatment on immediate postoperative graft function.
Methods. A model of acute double-lung transplantation in rats was used to assess graft function. Transplanted controls after 2 (group I) and 16 hours of ischemia (group II) were compared to a recipient (group III; 16-hour ischemia) and a donor treatment group (group IV; 16-hour ischemia) using the lazeroid U74389G (6 mg/kg). Serial assessment of alveolar–arterial oxygen difference, dynamic lung compliance, airway and pulmonary vascular resistance was obtained during a 2-hour reperfusion period. Final analysis included survival, weight gain, and histologic examination.
Results. Graft function was significantly better after 2 hours of ischemia than in any of the three 16-hour ischemia groups (II, III, IV). After 16 hours of ischemia, donor treatment provided superior graft function with respect to dynamic lung compliance, airway resistance, and alveolar–arterial oxygen difference when compared with groups II and III. The pulmonary vascular resistance was significantly higher in group III when compared with groups II and IV. Graft weight increase reflecting edema was highest in groups III (104%) and II (98%).
Conclusions. After prolonged ischemia only donor treatment with the lazeroid U74389G was able to significantly reduce ischemia–reperfusion-related graft dysfunction.
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Introduction
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IIschemia– reperfusion injury is the leading cause of early graft dysfunction after clinical lung transplantation [1]. The delicate architecture of the lung and the detrimental effects of permeability damage in the lung account for its high susceptibility to delayed graft function. Cell swelling, interstitial and intraalveolar edema inhibit oxygen transfer from the alveoli to the blood [2]. In addition, surfactant production and function may be severely impaired because of alveolar type II cell injury and deactivation of intraalveolar surfactant by permeated proteins [3–5]. Therefore, hypoxemia, increased vascular resistance, and reduced graft compliance are considered hallmarks of ischemia–reperfusion injury [6]. Once the inflammatory cascade has been triggered, therapeutic interventions have often proven futile [1].
Oxygen-free radical formation and release of proteases by polymorphonucleated neutrophils and endothelial cells are considered one of the major mechanism by which lung injury occurs in ischemia–reperfusion injury [7, 8]. More than three times the total number of circulating polymorphonucleated neutrophils are physiologically marginated and resident in the lung [9]. This pool of neutrophils increases further during endothelial cell and polymorphonucleated neutrophil activation [10]. Therefore, resident neutrophils in the donor lung represent a significant inflammatory potential during ischemia [11].
The use of antioxidants has proven beneficial in various experimental settings for amelioration of ischemia–reperfusion injury [12–15]. In these studies antioxidants have generally been administered to the recipient only or as a combined form of treatment to donor and recipient animals. The impact of timing of drug administration on graft protection remains to be defined. The following study was conducted to test the hypothesis that donor pretreatment alone can provide superior graft function when compared to recipient treatment. This study addresses the importance of treating the resident neutrophil pool in the donor lung as well as preventing lipid peroxidation through oxygen-free radicals liberated during ischemia from donor endothelial cells. The study was conducted in an in vivo, acute double-lung transplantation model in the rat.
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Material and Methods
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Male Lewis rats (350 to 400 g) were obtained from Charles River, Salzfeld, Germany. All animals received humane care in compliance with the "Principals of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 85-23, revised 1985).
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Experimental Groups
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Each group consisted of 10 animals each. In all study groups the lungs were flush perfused and stored with a low-potassium dextran solution (Perfadex) at 4°C. The perfusion pressure was 20 mm Hg and the perfusate volume 60 mL/kg. On the basis of previous results the grafts were kept inflated during storage with an intratracheal pressure of 26 cm H2O [3].
Control animals received implants after 2 and 16 hours of cold ischemia (groups I and II, respectively). In group III, the lazeroid U74389G was given intravenously at 6 mg/kg to the recipient animal 30 minutes before graft reperfusion. In group IV the donor was pretreated with U74389G at 6 mg/kg given intravenously 30 minutes before flush perfusion. The dosage of 6 mg/kg was derived from previous studies by Sasaki [12] and Tanoue [13] and their colleagues. After intravenous application the initial half-life of U74389G is 90 to 120 minutes.
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Experimental Procedure
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The model of acute in vivo double-lung transplantation in the rat has been described in detail in a previous publication [16]. Figure 1
is a graphic illustration of the model design. At the end of flush perfusion the main pulmonary trunk was dissected from the right ventricle and the mitral valve of the graft closed with an 8-0 Prolene (Ethicon, Somerville, NJ) suture.
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Fig 1. . Model design. Double-lung block is implanted through custom-designed stents to the vessels of the left hilum of the recipient. Donor graft is ventilated through a separate ventilator. Serial assessment includes donor-specific oxygenation, graft vascular resistance, dynamic lung compliance, and airway resistance. (A/D = analog-to-digital; LAP = left atrial pressure; P.art. = pulmonary artery; PAP = pulmonary artery pressure; PC = personal computer; P.ven. = pulmonary vein.)
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For implantation into a syngeneic recipient the donor pulmonary trunk and left atrial appendage were connected to the left pulmonary artery and vein of the recipient rat using two custom designed T-shaped stents. The donor lungs were then placed partially within the left hemithorax of the recipient. One of these stents contained a custom designed Doppler flow probe (H-2R probe; Transonic Systems Inc, New York, NY) for blood flow measurement. Both stents were designed with a side port for blood retrieval and pressure measurements. The donor trachea was intubated with a 13-gauge cannula and the graft ventilated with a Harvard volume-controlled respirator (Harvard Rodent Ventilator model 683; South Natick, MA) at 14 mL/kg tidal volume and a positive end-expiratory pressure of 3 cm H2O. The inspiratory oxygen concentration was 1.0. After administration of heparin, blood reperfusion was initiated. The respiratory rate of the donor lung was adjusted to maintain a left pulmonary venous partial pressure of carbon dioxide of 30 to 40 mm Hg. Fluid sequestration and evaporation was replaced with either blood or crystalloid fluid to ensure a mean pulmonary artery pressure of 20 mm Hg and a hematocrit of 30% to 40%. The donor lung was kept moist by intermittent topical application of warm fluid. The pH measured in the pulmonary venous blood was titrated with 8.5% NaHC03 to achieve a pH of 7.25 to 7.5.
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Measurements
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The model design allowed serial assessment of pulmonary venous and arterial pressures and respective blood gases. In addition, online measurement of isolated graft blood flow, dynamic compliance, and resistance to airflow was performed every 20 minutes. On the basis of these data the alveolar–arterial oxygen difference and the pulmonary vascular resistance (PVR) were calculated. Each measurement was preceded by a single sigh ventilation and removal of edematous fluid or secretions. The duration of reperfusion was limited to 120 minutes. Final assessment included weight gain of the graft and standard histology.
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Bronchoalveolar Lavage
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Bronchoalveolar lavage was performed at the end of the reperfusion period. The trachea was intubated with a 13-gauge cannula and 3 mL of 4°C saline solution was infused by gravity at a rate of 10 mL/min. Lavage was repeated five times. The bronchoalveolar lavage was then centrifuged at 270 g and the cell-free supernatant frozen at -80°C.
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Phospholipid and Protein Determination in the Bronchoalveolar Lavage
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The surfactant pellet was resuspended in 154 mmol/L saline supplemented with 1.5 mmol/L calcium chloride. After separation of pellet and supernatant at 27,000 g for 30 minutes, the phospholipid content was determined from a 5-µL aliquot of both pellet and supernatant according to the method of Bartlett [17]. Protein levels were determined according to the method described by Lowry and colleagues [18] and levels were expressed in milligrams per milliliters.
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Histologic Examination
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At the end of the reperfusion period the left pulmonary lobe was dissected, flushed, and stored in formalin and then cut and stained with hematoxylin and eosin. A semiquantitative scale was used for evaluation, scoring the degree of interstitial and intraalveolar edema, extravascular granulocyte infiltration, and pulmonary hemorrhage (score: 1 = no, 2 = mild, 3 = moderate, 4 = severe).
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Statistical Analysis
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Data were analyzed with the Statistical Program for Social Sciences (SPSS for Windows Version 6.1.3, Birmingham, AL). All data are expressed as mean ± standard error of the mean. Analysis of continuous data, such as compliance, airway resistance, alveolar–arterial oxygen difference, ventilation–perfusion shunt, and PVR was performed by repeated-measures analysis of variance (ANOVA). The model used incorporated a fixed time effect, a fixed group effect, a time by group interaction effect, and a random animal effect. In addition, the change over time was statistically analyzed with this model. For multiple comparisons the Bonferroni adjustment was incorporated. Continuous data without repeated measurements, such as donor and recipient animals' weights, weight increase of graft, donor compliance, resistance and animal survival, as well as nonparametric data, such as the histologic semiquantitative analysis, were compared with the Mann-Whitney U test.
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Results
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Demographic and pretransplantation data are presented in Table 1. The four study groups were comparable in terms of donor and recipient weight (see Table 1). The type of pretreatment did not affect dynamic compliance or airway resistance, which was assessed in the donor animal immediately before graft perfusion (see Table 1). Treatment with U74389G and the timing of its administration had a significant impact on animal survival and percent graft weight gain. Recipient treatment with U74389G before reperfusion adversely affected survival in comparison to untreated controls (groups I and II) and donor treatment animals (group IV). The weight increase reflecting pulmonary hemorrhage and edema was significantly higher in group III in comparison to groups I and IV (p < 0.01; p < 0.05, respectively). The average amount of fluid suctioned from the endotracheal tube was 0.2 ± 0.1 mL per measurement in control group I, 0.81 ± 0.2 mL in control group II, 0.69 ± 0.1 mL per measurement in the recipient treatment group (group III), and 0.2 ± 0.1 mL per measurement in the donor treatment group (group IV). Comparing data obtained after 16 hours of ischemia, the difference between groups II and IV, as well as between groups III and IV, was significant (Mann-Whitney U test, p < 0.005 and p < 0.0006, respectively).
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Serial Measurements
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Figure 2 depicts the dynamic lung compliance of transplanted grafts during the reperfusion period. Extended ischemia significantly reduced the dynamic lung compliance in groups II, III, and IV compared with group I (repeated-measures ANOVA group I versus group II p < 0.0001; group I versus group III p < 0.0001; group I versus group IV p < 0.0005). Donor pretreatment resulted in a significantly higher dynamic lung compliance when compared with untreated controls after 16 hours of ischemia (group II) and recipient treatment animals (ANOVA p < 0.045; p < 0.034, respectively). The decrease in dynamic lung compliance in group III was significant by one-way ANOVA analysis (p < 0.004). The airway resistance (Fig 3) was significantly lower in groups I and IV than in groups II and III (group I versus group II p < 0.0001; group I versus group III p < 0.0001, group IV versus group II p < 0.0001; group IV versus group III p < 0.05). Recipient treatment also improved the airway resistance of group III in comparison with untreated controls (group II; p < 0.03).
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Fig 2. . Serial measurements of dynamic lung compliance. Donor pretreatment significantly improved graft compliance during reperfusion. (Repeated-measures analysis of variance: groups I/II p < 0.0001; groups I/III p < 0.0001; groups I/IV p < 0.0001; groups II/IV p < 0.045; groups III/IV p < 0.035.)
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Fig 3. . Airway resistance, measured in cm H20 • L-1 • s-1, is significantly lower if the donor animal was pretreated with the lazeroid U74389G when compared with untreated controls and recipient treatment. (Repeated-measures analysis of variance: groups I/II p < 0.0001; groups I/III p < 0.0001; groups II/IV p < 0.0001; groups II/III p < 0.03; groups III/IV p < 0.05.)
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The alveolar–arterial oxygen difference increased significantly over the duration of the study in all three 16-hour ischemia groups (one-way ANOVA: group II p < 0.00001; group III p < 0.00002; group IV p < 0.00001; Fig 4). Again, the shorter ischemic interval in group I provided superior function with respect to oxygenation when compared with the 16-hour ischemia groups. The alveolar–arterial oxygen difference was lowest in group IV (ANOVA group IV versus group II p < 0.0001; group IV versus group III p < 0.001).
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Fig 4. . Comparison of alveolar–arterial oxygen difference. In untreated controls and the recipient treatment group the alveolar–arterial oxygen difference was significantly higher than in donor pretreated animals. (Repeated-measures analysis of variance: groups I/II p < 0.0001; groups I/III p < 0.0001; groups I/IV p < 0.0001; groups II/IV p < 0.0001; groups III/IV p < 0.001.)
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The timing of administration of the lazeroid U74389G had a significant effect on the PVR (Fig 5). Recipient treatment resulted in a significantly elevated PVR with minimal blood flow through the graft at the onset of reperfusion. After the first 80 minutes of reperfusion the PVR in this group decreased slightly; however, the PVR was at all times significantly higher than both the 16-hour control and donor treatment groups (repeated-measures ANOVA p < 0.03; p < 0.04, respectively).
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Fig 5. . Resistance to pulmonary blood flow measured in mm Hg • mL-1 • min-1 is markedly elevated in the recipient treatment group with minimal blood flow at the onset of reperfusion. (Repeated-measures analysis of variance: groups I/II p < 0.0001; groups I/III p < 0.0001; groups I/IV p < 0.0001; groups II/III p < 0.03; groups III/IV p < 0.04.)
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Phospholipid Aggregates and Protein Levels in Bronchoalveolar Lavage
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The analysis of phospholipids obtained from the bronchoalveolar lavage in the four study groups are depicted in Table 2. Short ischemia is rewarded with higher fraction of surface active phospholipid aggregates and lower intraalveolar protein content. After 16 hours of ischemia, the total phospholipid levels were significantly higher in both treatment groups than in the untreated controls. The percentage of large phospholipid aggregates was similar in groups II, III, and IV. The protein levels in the lavage were significantly higher in the lazeroid treatment groups in comparison with untreated controls (see Table 2).
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Histology
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The results of the semiquantitative evaluation of the specimens are listed in Table 3. In the statistical analysis the amount of interstitial edema, intraalveolar edema, or extravascular granulocyte infiltration was similar in all four study groups. The degree of intrapulmonary hemorrhage was significantly higher in group III animals when compared with group IV (Mann-Whitney U test; p < 0.05).
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Comment
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Various experimental studies have shown a beneficial effect of antioxidants for amelioration of ischemia–reperfusion injury [12–15]. Lazeroids are 21-aminosteroids, which are known to be potent inhibitors of iron-catalyzed lipid peroxidation [19–21]. In an isolated rat lung perfusion model, Sasaki and colleagues [12] have recently compared the impact of treatment with the lazeroid U74389G using combined donor and recipient treatment to untreated controls. According to their results the combined treatment significantly improved oxygenation and reduced airway resistance during 2 hours of reperfusion. Limitations of this study include the lack of interaction of an intact recipient immune system with the isograft in this in vitro model. In addition, it remains unclear whether donor or recipient treatment is more advantageous. Tanoue and colleagues [13] evaluated a similar lazeroid (U74500A) in a canine acute lung transplantation model. Timing of drug application was similar to the previously mentioned study in that one group received a combination of both donor and recipient treatment and was compared to untreated controls. Again, the lazeroid treatment provided significantly better graft function in this in situ and in vivo model. Oxygenation was improved and PVR was decreased after lazeroid administration. As with the Sasaki study, this study did not address the question of donor versus recipient treatment with an antioxidant.
The use of U74389G in the present study for donor pretreatment resulted in superior graft function with respect to dynamic lung compliance, airway resistance, and alveolar–arterial oxygen difference in comparison with recipient treatment and untreated controls after 16 hours of cold graft ischemia. In addition, the lungs of donor pretreated animals showed the least amount of weight gain reflecting graft edema and significantly less pulmonary hemorrhage. Although better function was achieved, the results obtained after donor treatment were still significantly worse than in isograft with short ischemia. In this study recipient treatment resulted in a significant increase in PVR even in comparison to untreated 16-hour controls. This certainly implies that delaying lazeroid treatment can adversely affect function of the transplanted grafts.
The superior protection from ischemia–reperfusion injury with donor lazeroid treatment may be explained by the prophylactic treatment of marginated neutrophils and endothelial cells in the donor lung. Both cell types are considered the two major sources of oxygen-free radicals in the lung. In a histologic study performed by Pickford and colleagues [22], prolonged ischemia without reperfusion was shown to result in endothelial blebbing and widening of the basement membrane. Even during hypothermic storage iron- and calcium-mediated free radical production were considered to be an important mechanisms for oxidative damage of lung tissue [8]. This is especially true in the lung with its oxygen reserves in the inflated alveoli. In comparison with other solid organs, the lung is unique in that most of the marginated neutrophils are located in precapillary and capillary parenchymal vessels. This is in sharp contrast to most other organs, where neutrophil margination can be found mainly in the postcapillary venules [23]. In addition, the cross-sectional area of neutrophils changes with its momentary location in the lung [10]. It is important to note that the largest cross-sectional area of neutrophils is found in the capillary bed. More than 35% of the capillary segments of the lung require neutrophil deformation to allow passage [24]. As activation results in a further increase in polymorphonucleated neutrophil cross-sectional area [10] and the decrease in temperature during flush perfusion reduces the ability of the neutrophils to deform to pass through capillaries, antegrade organ flush perfusion fails to eliminate the resident neutrophils from the donor lung. Injury to the cell membranes of neutrophils and endothelial cells then allows leakage of oxygen-free radicals and proteases during cold ischemic storage. The initial tissue injury is then further propagated during reperfusion, when endothelial cell swelling, intravascular coagulation, and cell debris significantly reduce flow through the vascular lumen [25]. Additional damage may then be attributable to subsequent recipient neutrophil activation, margination, sequestration, and emigration.
Preventing lipid peroxidation with antioxidants dissolved into the intravasum, interstitial, and intracellular compartments therefore limits the amount of tissue damage during the ischemic and reperfusion phase. At the time recipient treatment was initiated in this study the inflammatory cascade may have already been triggered and therefore, antioxidant treatment was not sufficient to limit tissue damage.
The significantly elevated PVR in recipient treatment when compared to untreated controls cannot be merely explained with possible negative inotropic effects of the drug or its solvents in rats, as this elevated PVR coincides with morphologic injury as seen in the histologic section of the donor lung. Therefore, it seems likely that the delayed administration of the lazeroid may accentuate the injury of endothelial cells and polymorphonucleated neutrophils.
The general limitations of a small animal study with respect to transferring results to clinical transplantation certainly apply to this study. This experiment uses healthy and uninjured lung donors. This differs substantially from clinical reality, where the inflammatory cascade may already be ignited many hours before organ harvesting. Barotrauma, oxygen toxicity, repeated suctioning of the airways, infection, mechanical tissue damage, fluid overload, and hormone interference can all result in gray injury. Lipid peroxidation through oxygen-free radicals may be one of the mechanism involved.
Therefore, donor treatment with drugs, such as the 21-aminosteroid used in this study, may require repeated administration very early after hospital admission to prevent or minimize organ damage. This may increase the number of suitable donor organs.
Limiting antioxidant treatment to the donor can avoid the possible negative adverse effects during recipient treatment, which include the potential for bacterial or fungal infection.
To conclude, donor pretreatment with the lazeroid U74389G can provide superior protection from ischemia–reperfusion injury in this in vivo rat double-lung transplantation model. Recipient treatment before graft implantation with U74389G in this model may result in a further decline of organ function during reperfusion.
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Acknowledgments
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We thank Ingrid Schmidt-Richter from the Department of Cardiothoracic Surgery of the Hannover Medical School in Hannover, Germany, for the phospholipid analysis. Dr Hausen was supported by the German Research Society.
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Footnotes
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Address reprints requests to Dr Hausen, Transplantation Immunology, Department of Cardiothoracic Surgery, Stanford University Medical Center, Falk CVRB, 300 Pasteur Dr, Stanford, CA 94305-5247.
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References
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Abstract
Introduction
