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Ann Thorac Surg 1995;60:1376-1381
© 1995 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine, St. Louis, Missouri
Accepted for publication June 22, 1995.
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. Using an isolated ex vivo rabbit lung model, we studied the effects of preservation temperature on pulmonary capillary filtration coefficient (Kf) and lipid peroxidation in rabbit lungs inflated with 100% O2 after preservation with or without the O2 free radical scavenger dimethylthiourea. New Zealand white rabbits weighing 2.7 to 3.1 kg were intubated and ventilated with room air or 100% O2 (tidal volume = 25 mL). After heparinization and sternotomy, the pulmonary artery was flushed with low-potassium–dextran–1% glucose solution (200 mL). The heart-lung block was excised, submerged, and stored for 24 hours at 1° or 10°C. After 24-hour preservation, the heart-lung block was suspended from a strain-gauge force transducer and ventilated with room air. The pulmonary artery cannula was connected to a reservoir of hetastarch solution. The lungs were flushed briefly with the hetastarch solution, and the reservoir was raised sequentially at 8-minute intervals to achieve 1.0 to 1.5 mm Hg increments in pulmonary artery pressure. Lung weight gain, airway pressure, pulmonary artery pressure, and left atrial pressure were measured continuously. The slope of steady-state lung weight gain was used to determine Kf (g • min-1 • cm H2O-1 • 100 g-1 wet weight).
Results. Twenty-four–hour lung preservation at both 1° and 10°C increased Kf. A similar increase in Kf was observed in lungs stored at 1°C while inflated with 100% O2. However, a significant increase in Kf was observed when lungs inflated with 100% O2 were stored at 10°C. This increase in Kf was ameliorated by dimethylthiourea. Thiobarbituric acid–reactive substance levels were increased in lungs stored at 10°C while inflated with 100% O2. This finding was eliminated by dimethylthiourea.
Conclusions. These results indicate that free radical injury occurs during the ischemic phase when lungs are stored at moderate hypothermia while inflated with 100% O2.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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To evaluate this possibility, we determined the effects of preservation temperature on pulmonary capillary filtration coefficient (Kf) and lipid peroxidation of rabbit lung inflated with 100% O2 after preservation with or without the O2 free radical scavenger dimethylthiourea (DMTU).
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Material and Methods |
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THIOBARBITURIC ACID–REACTIVE SUBSTANCE ASSAY.
Postpreservation lung tissue thiobarbituric acid–reactive substance (TBARS) was measured in another series of experiments in which Kf was not determined. In a control group (n = 5), TBARS levels were measured immediately after LPDG flush. After 8-hour and 24-hour preservation at 10°C, TBARS levels of the lungs stored at RA (n = 5) and 100% O2 (n = 5) were measured. In lungs preserved at 1°C, TBARS levels were measured after 24-hour preservation (RA, n = 5; 100% O2, n = 5).
Excision and Preparation of Preserved Lungs
The preparation of the heart-lung block has been described previously [11, 12]. Briefly, New Zealand white rabbits free from respiratory infections and weighing 2.7 to 3.1 kg were used. Rabbits were premedicated with subcutaneous administration of atropine sulfate (0.25 mg/kg), ketamine hydrochloride (35 mg/kg), and acepromazine maleate (0.6 mg/kg) and anesthetized with intravenous administration of sodium thiopental (25 mg/kg). Minimal supplementary doses of thiopental were given when required. The animals were heparinized (700 IU/kg).
An endotracheal tube was introduced through a cervical tracheostomy, and the animals were connected to a mechanical ventilator (model 671; Harvard Apparatus Co, Millis, MA) (tidal volume, 25 mL; rate, 30 breaths/min; positive end-expiratory pressure, 0.5 cm H2O). After median sternotomy, the bilateral superior venae cavae, the inferior vena cava, the ascending aorta, and the main pulmonary artery were dissected free and loosely encircled with individual ligatures. A catheter 2.2 mm in diameter (No. K50L; Baxter Healthcare Co, Valencia, CA) was introduced into the main pulmonary artery through the right ventricular outflow tract and secured in place. Cardiac inflow occlusion was accomplished by ligation of all three venae cavae, and the pulmonary artery was ligated around the cannula. The left atrial appendage was amputated, and the pulmonary artery was flushed with LPDG solution [7].
All lungs were flushed with 200 mL of LPDG by gravity from a height of 60 cm above the chest. The extracellular solution LPDG was used for two reasons. First, intracellular solutions (eg, Euro-Collins solution, University of Wisconsin solution) produce intense vasoconstriction in untreated rabbit lungs, thereby preventing satisfactory flush for storage purposes. Second, our laboratory has great experience with the use of LPDG solution in a variety of animal models [10, 12, 13].
After completion of the flush, a 19-gauge tube (model 88ID9F18; Terumo, Tokyo, Japan) was inserted through the amputated left atrium, and the heart, lungs, and esophagus were extracted en bloc from the thorax with utmost care to prevent lung injury from atelectasis, disruption of lung surface, or any manipulation of lung tissue. The lungs were ventilated continuously during this procedure. The lung was inflated at end-tidal volume, and the tracheal tube was clamped. A glass rod was then inserted through the lumen of the esophagus for subsequent suspension of the specimen. The heart-lung block was completely immersed in saline solution and covered with a plastic sheet. The temperature of the saline solution was maintained at the preservation temperature.
Estimation of Kf
The assessment technique was the same for all groups. The heart-lung block was placed in a constant-temperature (37°C) and humidified Plexiglas box. The block was suspended from a strain-gauge force transducer (model FT03; Grass Instruments, Quincy, MA) attached to a recorder (model 2600; Gould, Cleveland, OH), which was calibrated full scale from 0 to 10 g by means of a bridge amplifier (Accudata 143; Honeywell, Denver, CO). The lungs were ventilated with RA (tidal volume, 25 mL; rate, 30 breaths/min; positive end-expiratory pressure, 0.5 cm H2O). Before the assessment, the lungs were flushed with 30 mL of room-temperature hetastarch solution (6% hetastarch solution with 0.9% NaCl; Du Pont Pharmaceuticals, Wilmington, DE) in which the pH was adjusted to 7.35 to 7.45 before each experiment. The osmolarity of the hetastarch solution was 310 mosmol/L. Flushing pressure was kept 1 to 2 mm Hg higher than the peak inspiratory pressure. Flushing was completed within 5 minutes.
After flushing, the left atrial cannula was attached to a pressure transducer, and the pulmonary artery cannula was attached to a suspended reservoir of hetastarch solution. Changes in pulmonary artery pressure (PAP), left atrial pressure, airway pressure, and total weight of the preparation were measured continuously (Accudata 143) and recorded (Gould model 2600) during the assessment. Prior to the measurement of Kf, the lungs were hyperinflated twice to remove any area of atelectasis.
Increases in PAP and resultant lung weight were accomplished by elevation of the reservoir. First, PAP was raised to airway pressure and then increased to 17 cm H2O in increments of 1.0 to 1.5 cm H2O at 8-minute intervals. After the reservoir was elevated, rapid weight gain was observed within the first 2 minutes. This rapid gain was primarily the result of increased endovascular and intracardiac volume caused by the increased hydrostatic pressure. This phase was followed by a slower rate of weight gain, which we interpreted as filtration of fluid out of the microvasculature into the lung interstitium. Left atrial pressure gradually increased to reach the same level as PAP (PAP = left atrial pressure = pulmonary capillary pressure) during the first 2 to 5 minutes, and after this period, the rate of weight gain became stable. The rate of weight gain over the last 3 minutes of each 8-minute interval was plotted against pressure to determine the Kf.
Calculation of Kf
The rate of weight gain was plotted as a function of pulmonary capillary pressure. A two-variable linear regression was used to obtain Kf as the slope of the line relating the weight gain rate and pulmonary capillary pressure. At PAP greater than 15 cm H2O, acceleration in the rate of weight gain was sometimes observed, and a steady-state left atrial pressure (PAP = left atrial pressure) could not be achieved. These points were excluded when a curve became nonlinear (correlation coefficient < 0.96). Under these conditions, the initial three to five points were used to calculate Kf in each case. The Kf was expressed as grams per minute per centimeter of water per 100 grams of wet lung weight.
Wet lung weight was calculated as follows: WLW = TW - RW, where WLW was wet lung weight, TW was total weight of the preparation before assessment, and RW was the weight of the remaining tissue after the lungs were removed from the preparation after assessment.
TBARS Assay
For these experiments, the heart-lung block was excised and preserved as for the Kf experiments. After completion of the preservation period, the lower lobes were rapidly frozen in liquid nitrogen. A portion of the frozen lung was weighed (wet weight) and dried in an oven at 60°C to constant weight (dry weight), and wet to dry weight ratios were calculated. The remainder of the lung tissue was kept at -70°C for measurement of TBARS levels.
The TBARS levels were measured according to the method of Ohkawa and colleagues [14] with 10% weight per volume homogenate. Aliquots (0.2 mL) of this homogenate were added to tubes containing 0.2 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid solution adjusted to 3.5 pH with NaOH, and 1.5 mL of 0.8% solution of thiobarbituric acid. The mixture was brought to a volume of 4 mL by the addition of distilled water, heated at 95°C for 60 minutes, and then cooled. One milliliter of water and 5 mL of butanol/pyridine (15:1, vol/vol) were added. The solution was centrifuged at 4,000 rpm for 10 minutes. The fluorescent intensity of the top layer was read in a spectrophotofluorometer (model U-3210, excitation of 515 nm and emission of 553 nm; Hitachi Instrument Inc, Danbury, CT). The TBARS levels were determined by reference to a standard curve created with 0.1, 0.5, 1.0, and 2.0 nmol of 1,1,3,3-tetramethoxypropane (Sigma), and the results were expressed as nanomoles of 1,1,3,3-tetramethoxypropane per gram of dry lung.
Statistical Methods
The results are presented as the mean ± the standard error of mean. Statistical analysis was performed using analysis of variance. Significance was accepted at the 95% confidence level (p < 0.05).
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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), there were no significant differences in Kf between lungs inflated with RA (0.463 ± 0.019) or 100% O2 (0.515 ± 0.032). Further, DMTU had no significant effect on Kf in lungs inflated with RA (0.436 ± 0.043) or 100% O2 (0.531 ± 0.049). However, 10°C preservation significantly increased Kf of lungs inflated with 100% O2 (1.244 ± 0.130) after 24-hour preservation (Fig 1B). This increase was not observed in the similar group treated with DMTU (Kf = 0.560 ± 2.050). Room air inflation, however, did not increase Kf (0.560 ± 0.026) in the 10°C preservation group, nor was Kf affected by DMTU in this group (Kf = 0.518 ± 0.032).
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).
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). After 24-hour preservation, a further decrease (p < 0.001) in TBARS levels was observed in the lungs inflated with RA (47.1 ± 8.3). On the other hand, TBARS levels in lungs inflated for 24 hours with 100% O2 (82.2 ± 6.6) were significantly higher than those observed in lungs inflated with RA (p < 0.001).
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). There was no significant difference between these groups. Dimethylthiourea had no effect on the TBARS level of lungs preserved at 1°C. On the other hand, DMTU selectively suppressed the TBARS levels of lungs inflated with 100% O2 during 10°C preservation (Fig 3B).
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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However, it is important to note that in the lung, unlike other organs, ischemia does not necessarily result in tissue hypoxia when intraalveolar O2 concentration is maintained. In fact, it is possible that lung injury by toxic O2 metabolites occurs even during ischemia. Fisher and associates [9] demonstrated that reperfusion was not necessary for lipid peroxidation during ischemic insult of the ventilated rat lung, and elevated FiO2 during ischemia accelerated the rate of tissue injury. Becker and co-workers [4] showed that warm ischemia with 95% O2 inflation significantly increased vascular permeability of the ferret lung, and subsequent reperfusion did not make further changes in permeability. Although these results suggest that a high FIO2 may cause lung injury during warm ischemia, it is not clear whether O2 free radicals are produced during the ischemic phase under storage conditions used in clinical lung transplantation.
In the current study, the toxic effect of 100% O2 inflation on Kf was demonstrated when lungs were kept at 10°C for 24 hours. These data are consistent with those from a previous study from our laboratory [10]. The change caused by 100% O2 inflation was selectively improved by the addition of DMTU to the pulmonary flush solution. These observations suggest that static inflation with 100% O2 may facilitate production of toxic O2 metabolites primarily during ischemia at moderate hypothermia and result in lung injury that is manifested by increased pulmonary vascular permeability after 10°C preservation for 24 hours.
In contrast with the observation at 10°C preservation, the lungs inflated with 100% O2 during 1°C preservation had no increase in Kf. It has previously been demonstrated that the peak levels of O2 free radical production from polymorphonuclear leukocytes was at 37°C and that production was reduced with decreasing temperature [16]. These data suggest the possibility that the different effects of 100% O2 inflation on Kf between 1° and 10°C storage may derive from the differences in the production of O2 free radicals at each preservation temperature.
Lipid peroxidation was evaluated by the measurement of lung tissue TBARS levels, which represent in large part the final stage of decomposition of peroxidized lipids [17]. The TBARS assay is simple and sensitive. Many studies have demonstrated that lung TBARS levels increase after ischemia-reperfusion [18, 19]. The current experiments, however, showed that preservation at both 1° and 10°C decreased TBARS levels of lung tissue in relation to ischemic time. Our data are compatible with the findings in a previous study of rat lung [20], in which 6-hour and 12-hour preservation (0°C) after initial flush decreased TBARS levels of statically inflated lungs. In contrast, Fisher and colleagues [9] demonstrated that ischemia increased TBARS values in rat lungs in relation to FiO2 and ischemic time. In that study, however, isolated rat lungs were ventilated during warm ischemia. The differences in TBARS levels during ischemia may be attributable to differences in preservation temperature and ventilation during ischemia.
For 10°C preservation periods of 8 hours, there were no significant differences in TBARS levels between RA and 100% O2 inflation. Our previous study [10] demonstrated changes in Kf were not influenced by differences in intraalveolar O2 concentration when the 10°C preservation period was within 8 hours. Both results indicate that lung injury with 100% O2 inflation is not critical within an 8-hour preservation period at 10°C.
As was observed in the Kf experiments, there was no significant difference in TBARS levels between inflation with RA or 100% O2 after 24-hour preservation at 1°C. In contrast, at 10°C preservation, TBARS levels of lungs inflated with 100% O2 were significantly higher than those of lungs inflated with RA. The difference caused by 100% O2 inflation was ameliorated by DMTU. It may be questioned that this small but significant difference in TBARS levels reflects a toxic effect of 100% O2 inflation. However, DMTU improved the deterioration of both Kf and TBARS simultaneously. We therefore believe that this difference in TBARS levels was caused by the toxic effect of 100% O2 inflation during storage.
In the current study, we used the oxygen free radical scavenger DMTU, which has a low molecular weight and directly nonenzymatically counteracts hydrogen peroxide and hydroxy radicals [21, 22]. Many studies have demonstrated that DMTU is effective in ameliorating lung ischemia-reperfusion injury. Recently, Lambert and Egan [23] showed that canine lung allograft function after transplantation was adequately preserved after 12 hours of storage when DMTU was administered in the flush solution at the time of harvest. Dimethylthiourea infused at the time of implantation was less effective. In their study, however, canine lungs were inflated with 100% N2 and preserved at 4°C for 12 hours.
Although the current study demonstrated that 100% O2 inflation during moderate hypothermic storage caused lung injury likely due to oxygen free radicals, the mechanism is not clear. Seibert and associates [24] have shown that isolated rat lungs perfused with as much as 200 mL of buffer solution still contain a substantial number of leukocytes, and those leukocytes contribute to ischemia-reperfusion–induced microvascular injury. Therefore, it is possible that leukocytes resident in the allograft after flushing contribute to the formation of oxygen free radicals that cause microvascular injury. It has also been demonstrated that xanthine oxidase catalyzes superoxide generation within the endothelial cell itself from molecular oxygen, thereby producing direct endothelial injury independent of other mechanisms [25]. The precise mechanisms for microvascular injury during preservation remain to be determined.
In summary, static inflation with 100% O2 increased pulmonary vascular permeability and lipid peroxidation levels more than RA inflation without the effect of reperfusion. These lung injuries were observed only after 24-hour preservation at 10°C and were ameliorated by DMTU added to the prestorage pulmonary artery flush solution. We conclude that lung injury caused by O2 free radicals occurs during ischemia when lungs are preserved at moderate hypothermia inflated with 100% O2.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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We thank Mary Ann Kelly for the preparation of the manuscript and Richard B. Schuessler, PhD, for statistical advice.
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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