INTRODUCTION

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In solid organs such as the heart and liver, it is well known that the levels of energy compounds such as adenine nucleotides play an important role in maintaining organ functions during cold storage (1). The lung differs from other solid organs, because it has a low blood pressure system, its capillary vasculature is highly developed, and it contains air when inflated. So far, the level of energy consumption in the lung during warm ischemia (37° C) has been analyzed in detail using lung tissue slices and isolated perfused lungs, and it has been found that the pulmonary parenchyma requires oxidative metabolism to carry out a variety of functions such as protein synthesis and phospholipid synthesis (5). It has also been reported that since the aerobic metabolism was inhibited in deflated lungs, the extent of energy depletion was high, thus intensifying the ischemia-reperfusion (I-R) injury on warm ischemia (9, 10). However, changes in the energy level during cold ischemia according to alveolar state and temperature and their clinical significance are not clear (11). For example, according to Date and coworkers (13), adenosine triphosphate (ATP) levels in the rabbit lungs that had been inflated with room air at 10° C, were stable even after 24-h preservation. In contrast, Hall and coworkers (11) reported that the energy state in the rabbit lungs decreased to 75% of the baseline value at 4° C after 24-h preservation. Because pulmonary function after storage was not well documented and adenosine diphosphate (ADP) and adenosine monophosphate (AMP) levels in the lungs were not measured in these studies, the influence of the energy state on I-R injury remains uncertain. Some studies have indicated that the pulmonary functions were maintained most favorably when the lungs were stored in an inflated state at a normal tidal volume (VT) (15, 16), whereas other studies have shown that the pulmonary functions were better maintained when the lungs were stored in a hyperinflated condition (17, 18). Furthermore, Steen and coworkers (19) suggested that safe cold ischemic storage of the lungs can be done with the lung in an atelectatic state. There have also been conflicting reports on the relationship between the alveolar gases during storage and post-storage pulmonary functions, the most favorable results being obtained with 100% O₂ (20), room air (16), and 100% N₂ (21), whereas Van Raemdonck (24) reported that the type of gas did not affect the degree of I-R injury.

However, none of these studies on cold ischemia has analyzed the relationship between the energy level during storage according to alveolar conditions and the pulmonary function after reperfusion. If the influence of the deflated and anaerobic conditions during cold storage on lung injury can be clarified, the optimal condition of alveoli during lung storage will be suggested.

To determine the effect of inflation and oxidative metabolism on I-R injury, lungs were stored inflated, deflated, aerobically or anaerobically, and the pulmonary functions following cold storage were measured. In addition, to analyze the relationship between the pulmonary functions and energy level, changes in the level of high-energy phosphates, pyruvate, and lactate of lungs before and after storage and after reperfusion were assessed. Furthermore, to clarify alveolar cell injury during preservation, bronchoalveolar lavage fluid (BALF) after storage was analyzed. We employed a previously reported isolated rat lung perfusion model to measure post-storage pulmonary functions. In this model, homologous blood was used as perfusate, and venous blood that had been subjected to hypoxic ventilation using a biological deoxygenator, was perfused to a study lung (ventilated by 100% O₂) using a fixed flow pump. This model allows sensitive and reproducible assessment of the pulmonary functions after storage (25, 26).

	METHODS

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Donor Operation

Male Lewis rats, ranging in body weight from 250 to 300 g, were used. The rats were anesthetized with 30 mg/kg of pentobarbital, and after tracheotomy, endotracheal intubation was performed. During surgery, the lungs were ventilated using a Harvard ventilator (model 665) under the following conditions: room air, VT = 3.0 ml/body weight, 60 breaths/min, and no positive end-expiratory pressure (PEEP). After making a median abdominal incision and sternotomy, 400 U of heparin was administered into the abdominal aorta. Then, a no. 14 cannula was inserted into the pulmonary artery (PA). Just before performing PA flush, the abdominal aorta and the left auricle were severed to allow the pulmonary blood to flow freely. PA flush was performed from a height of 20 cm. The heart-lung block was then removed, and the right hilum was ligated using 1-0 silk.

Operation for Deoxygenator Lung

About 20 min before reperfusion, another group of Lewis rats was anesthetized in the same manner. After tracheotomy, endotracheal intubation was performed. During surgery, the lungs were ventilated using a Harvard ventilator (model 665) under the following conditions: room air, VT = 3.0 ml/body weight, 60 breaths/min, no PEEP. After making a median abdominal incision and sternotomy, 400 U of heparin was administered into the abdominal aorta. Then, a no. 14 cannula was inserted into the PA. The abdominal aorta and the left auricle were severed to allow a free flow of the pulmonary blood. The heart-lung block was then removed and placed in a perfusion apparatus within a chamber.

Perfusion Circuit

The perfusion circuit was started using 30 ml of fresh blood that was obtained from three rats (1,000 IU of heparin was administered to each rat). After cold storage of each heart-lung block, the right lung was severed, and the heart-left lung block was placed in a perfusion apparatus within a chamber (temperature: 37° C and humidity: 100%) (Figure 1). The blood was perfused from the venous blood reservoir via PA to the left lung using a double head roller pump. The blood from the left lung was collected in a blood reservoir and perfused to a deoxygenator lung using a pump. The perfusion rate was gradually increased to 4 ml/min within the first 10 min, and then the lung was perfused at 4 ml/min for 120 min at 37° C. The venous blood temperature was monitored throughout the experiment and kept at 37° C. Also, if the fluid in the tracheal tube exceeded the three-way valve because of pulmonary edema, the perfusion was stopped to prevent jeopardizing of the respirator's operation.

View larger version (24K): [in this window] [in a new window]   Figure 1.   Schema of the perfusion circuit.

Experimental Groups

There were five experimental groups, and in all groups, phosphate-buffered saline (PBS) was used for flushing and storage (Table 1). In the fresh group, each heart-left lung block was reperfused or used for BALF study immediately after flush (n = 6, each). In the room air (RA), deflated (DEF), and nitrogen (N₂) groups, each heart-left lung block was reperfused or BALF was collected (n = 6, each) after 6 h of cold storage. In the RA group and N₂ group, the lungs were inflated with room air or with nitrogen at 14 cm H₂O. In the DEF group, the lungs were deflated passively. In the N₂ group, to eliminate the dissolved oxygen from PBS, the solution was preequilibrated with 100% N₂ bubbling (2 L/min) for 60 min before flushing. The lung specimens in a Petri dish were transferred to sealed containers that had been stored at 4° C, and the containers were equilibrated with 100% N₂. In addition, the static lung compliance was measured after preservation in each group (n = 5, each). Furthermore, to investigate the changes in the energy level of the lungs that were stored at 37° C, the deflated lungs were stored for 6 h at 37° C (WDEF group, n = 6).

Physiological Measurements

Blood gases (pH, partial oxygen pressure in mixed venous blood [Pv_O2] and mixed venous CO₂ pressure [Pv_CO2]) in reservoir 1 were analyzed just before reperfusion (baseline) and after reperfusion (at 10, 20, 30, 40, 50, 60, 90, and 120 min), and the blood gases (pH, Pv_O2, and Pv_CO2) in the effluent blood from the left lung were measured to calculate the shunt rate. Moreover, throughout the study, the mean pulmonary arterial pressure () and the peak inspiratory pressure (PIP) were measured, and they were recorded at 10, 20, 30, 40, 50, 60, 90, and 120 min during reperfusion. At the end of reperfusion, the lower one-third of the left lung was weighed, and after drying for 72 h at 55° C, it was weighed again to calculate the wet-to-dry (W/D) ratio.

Metabolic Study

The right upper lobe after flushing and the right lower lobe after storage were taken, the samples were rapidly frozen by clamping between bronze tongs precooled at the temperature of liquid N₂, and the tissue was lyophilized overnight. The concentrations of lactate and pyruvate were measured from neutralized perchloric acid extracts of the lung using enzymatic methods (27).

The concentration of adenine nucleotides was measured in the right mediastinal lobe immediately after flushing, in the right middle lobe after storage, and in the upper one-third of the left lung after completion of reperfusion. Using the method of Cross and colleagues (28) to minimize the effect of the contamination of adenine nucleotides from blood components, the adenine nucleotides in the pulmonary blood were subtracted on measuring the concentration of adenine nucleotides in the lung tissue after reperfusion. The lung tissue was promptly clamped with bronze tongs that had been cooled in liquid nitrogen, and the clamped tissue was lyophilized overnight. When samples were taken from the left lung after 1 h of reperfusion, the pulmonary effluent blood was also collected, and the lung samples were frozen by immersion in liquid nitrogen, and lyophilized overnight. The concentrations of adenine nucleotides in the lung cells or in the blood were determined by a modification of the technique reported by Kamiike and coworkers (2). The levels of adenine nucleotides, ATP, ADP, and AMP, were measured by high-performance liquid chromatography on an anion exchanger column, DESE-2SW (4.6 × 250 mm) (Tosoh Co.), equilibrated with 380 mM sodium phosphate buffer (pH 6.0). The energy charge was defined as (ATP + 0.5ADP)/ (ATP + ADP + AMP).

The hemoglobin content of the lung homogenates was determined as described by Cross and colleagues (28). Regarding the reperfused samples, the corrected values were calculated as follows to eliminate the contamination of adenine nucleotides in intrapulmonary blood: lung adenine nucleotides = [adenine nucleotides in lung (+ intrapulmonary blood) homogenates]*  {adenine nucleotides in intrapulmonary blood: adenine nucleotides in blood perfusate × [hemoglobin content of lung (+ intrapulmonary blood) homogenates]/hemoglobin content of blood perfusate}.

BALF Study

BALF of the whole lungs was performed at the end of the preservation. A syringe containing 10 ml of warm saline (37° C) was set 15 cm H₂O above the preserved lungs, then the tracheal tube was connected to the syringe, allowing the saline to flow into the airway spontaneously. Then the lavage fluid was withdrawn very gently with a 10-ml syringe. This procedure was repeated 3 times. The lavage was immediately centrifuged at 500 × g for 10 min at 4° C and the supernatant was stored at 70° C until the biochemical analysis. Total protein was determined by the method of Lowry and associates (29). Phospholipids were measured by means of the choline oxidase assay reported by Ikuta and coworkers (30). The activity of lactate dehydrogenase (LDH) was measured by the method developed by Bergmeyer (27).

Measurement of the Static Pulmonary Compliance

The static pressure volume (PV) curve after lung preservation was measured as Muscedere and coworkers (31) previously described. In short, the lungs were allowed to deflate passively after preservation. The compliance was then measured by manually inflating the lung with room air, beginning with two 0.5-ml aliquots of air then with 1-ml increments until a transpulmonary pressure of 25 cm H₂O was achieved. The deflation loop of the PV curve was constructed by withdrawing air in the same manner. After each inflation/deflation, the pressure in the lung was allowed to stabilize for 10 s.

Statistical Analysis

The numerical values in the present study were expressed as mean ± SE. Analysis of Variance (ANOVA) and a Scheffe's multiple comparison test were used to compare the results of the four groups, and p values of less than 0.05 were considered significant.

All animals received humane care in compliance with the Principles 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 Science (NIH Publication No. 85-23, revised 1985).

	RESULTS

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In the present study, no experiments were excluded owing to technical errors, and 170 rats were used in total: 24 rats for deoxygenator lungs, 72 as bleeder rats, 24 as experimental group rats, 24 for the BALF study, 20 for measurement of the static lung compliance, and six rats for warm ischemia without reperfusion.

In each group, the pH, Pv_O2, and Pv_CO2 of the mixed venous blood during reperfusion were stable and within the normal physiological range.

Shunt Fraction

In the fresh, RA, and DEF groups, the necessary measurements were conducted throughout the entire 120 min after reperfusion (Figure 2a). However, in one of the rats in the N₂ group, measurements were stopped after 60 min of reperfusion because the ventilation of the study lung became difficult owing to a high liquid content in the endotracheal tube brought about by pulmonary edema. In the fresh and RA groups, the shunt fraction was very stable at 3.5% throughout the study. For 120 min after reperfusion, the shunt fraction of the DEF and N₂ groups was significantly higher than that of the RA and fresh groups.

View larger version (17K): [in this window] [in a new window]   Figure 2.   (a) Shunt fraction during reperfusion in preserved lungs of the fresh, RA, DEF, and N2 groups. (b) Peak inspiratory pressure during reperfusion in preserved lungs of the fresh, RA, DEF, and N2 groups. (c) Mean pulmonary artery pressure during reperfusion in preserved lungs of the fresh, RA, DEF, and N2 groups. Bars are means ± SE. *p < 0.05 versus the fresh and RA group.

Peak Inspiratory Pressure

After reperfusion, the PIP of the RA and fresh groups was stable and comparable (Figure 2b). On the other hand, PIP increased after reperfusion in the N₂ and DEF groups, and there were significant differences in PIP between these groups and the fresh and RA groups.

For 120 min, there were no significant differences in after reperfusion among the four groups (Figure 2c).

W/D Ratio

The W/D ratio in the DEF and N₂ groups was significantly higher than that in the fresh and RA groups (Figure 3).

View larger version (54K): [in this window] [in a new window]   Figure 3.   Wet to dry weight ratios in preserved lungs of the four groups. Bars are means ± SE. *p < 0.05 versus fresh and RA groups.

Lactate and Pyruvate Concentrations

There were no significant changes in the concentrations of lactate during flushing in any group, and there were no significant differences in it among the four groups (Table 2). The concentration of lactate and the lactate/pyruvate ratio after storage were significantly higher in the N₂ group than in the other three groups.

Adenine Nucleotides

There were no significant differences in the concentrations of ATP and total adenine nucleotides (TAN) during flushing among the four groups (Figure 4). The concentrations of ATP and TAN after cold storage did not decrease significantly, and there were no significant differences among the four groups. Nonetheless, in the WDEF group (6 h of warm storage at 37° C), the levels of TAN, ATP, and energy charge significantly decreased after storage, and they were significantly lower than those of the other groups (Figure 3). The concentrations of ATP and TAN after reperfusion were significantly lower in the DEF and N₂ groups than in the fresh and RA groups (Figure 3). Although there were no significant differences in the energy charge during flushing between any of the groups, it was significantly lower after storage in the N₂ group. There were no significant differences in the concentrations of TAN, ATP, ADP, and AMP in the blood among all groups, and the average intrapulmonary hemoglobin content was 0.30 ± 0.02 mg/dw mg.

View larger version (43K): [in this window] [in a new window]   Figure 4.   (a) Total adenine nucleotide in study lungs after flush, preservation, and reperfusion of fresh, RA, DEF, and N2 groups. (b) ATP in study lungs after flush, preservation, and reperfusion of fresh, RA, DEF, and N2 groups. (c) Energy charge in study lungs after flush, preservation, and reperfusion of fresh, RA, DEF, and N2 groups. Bars are means ± SE. *p < 0.05 versus the fresh and RA groups.

BALF Study

LDH activity and total protein in the BALF of the DEF group were significantly higher than those of the fresh, RA, and N₂ groups (Table 3). There were no significant differences among the fresh, RA, and N₂ groups.

Static Pulmonary Compliance

For the deflated lungs, there was a significant shift to the right in the composite PV curves after preservation, and a significant reduction in the lung volume at transpulmonary pressure of 5 to 25 cm H₂O (Figure 5).

View larger version (19K): [in this window] [in a new window]   Figure 5.   Pressure-volume curves after preservation in the fresh, RA, DEF, and N2 groups. *p < 0.05 compared with the other three groups.

	DISCUSSION

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As reported by other investigators (5), when deflated lungs were stored at 37° C, the concentration of high-energy phosphates significantly decreased in this study. However, the results of the present study showed that when deflated lungs were stored at 4° C, the concentration of high-energy phosphate did not decrease. The reason for this may be that (1) at lower temperatures, intrapulmonary energy was not consumed, or (2) intrapulmonary energy was consumed, but replenished. Nonetheless, when the lungs were stored anaerobically by inflating them with 100% N₂, the energy charge decreased and the concentration of lactate and lactate/pyruvate ratio increased, suggesting that the latter hypothesis is a more probable explanation. Consequently, energy will be consumed by the lung during cold storage, and anaerobic metabolism may not be sufficient to maintain the energy level. In this study, because we used glucose-free PBS as a preservation medium, the source of glycolysis would be glucose and glycogen from the lung. Accordingly, glucose-containing medium would have enhanced glycolysis and ATP production. However, this hypothesis does not seem probable because it has been shown that endogenous glycosyl units would be sufficient to support the lung anaerobic metabolism for over 22 h (13). Physiological data also showed that lungs that had been inflated with N₂ were in significantly worse condition than the lungs inflated with room air. These findings suggest that anaerobic metabolism alone can not maintain the pulmonary energy level during cold storage, and causes I-R injury. So far, several studies (21) have reported that ventilation with N₂ during ischemia protected the lungs against lipid peroxidation and pulmonary edema after reperfusion. The conditions of these experiments were different from those of this study because they were carried out at room temperature or 37° C, and lungs were ventilated during ischemia. It is well known that energy consumption varies according to tissue temperature and ventilation can change surfactant secretion and produce stress fractions of capillary endothelium, epithelium, and basement membrane (32). Although lipid peroxidation was not measured in our study and the previous studies did not examine energy metabolism during ischemia, the contradictory results between our study and the previous studies may be the result of differences in pulmonary energy depletion and enzyme activity such as phosphofructokinase, pyruvate kinase, and eicosanoid production (21) according to the tissue temperature and ventilation during ischemia. In the future, the relationships between concentratons of high-energy phosphates, lipid peroxidation, and tissue temperature during ischemia should be elucidated.

This study also demonstrated that the pulmonary functions after storage were significantly worse for deflated lungs than lungs that had been inflated with room air. Nonetheless, the energy level of the deflated lungs, such as TAN, ATP, ADP, AMP, and EC, after flushing and storage was comparable to that of the lungs in the RA and fresh groups, and unlike the lungs in the N₂ group, the concentration of lactate or lactate/ pyruvate ratio did not increase after storage. On the other hand, the DEF group showed significant decrease of TAN and ATP concentrations in the lung after reperfusion. It has been reported that decrease in the concentrations of TAN and ATP after reperfusion is caused by energy production disorders or by release of TAN and ATP resulting from destruction of the cells (2). In any case, decrease in the concentrations of TAN and ATP is caused by damage to the cell organs such as cell membranes or mitochondria. The BALF analyses, which showed significant increases in total protein and LDH activity in the DEF group after storage compared with the other three groups, supported the hypothesis of cellular injuries mentioned previously, because increases in LDH activity and total protein in BALF reflect injury of the alveolar cells and increased alveolocapillary membrane permeability (32, 33). These results indicate that deterioration in the functions of the lungs stored in a deflated condition resulted from physical damage to alveolar cells and alveolocapillary membranes. These findings are consistent with previous reports which found that hypoinflation increased pulmonary vascular permeability (16). Our study also showed that the static lung compliance after preservation was significantly lower in the deflated lungs compared with room air- and N₂-inflated lungs. Hausen and coworkers (18) reported that less inflation and longer ischemia resulted in a reduction of the large to small phospholipid aggregate ratio and deterioration of surfactant function in the bubble surfactometer, and Akashi and coworkers (9) suggested that inflation during ischemia would preserve the ability of cells to secrete pulmonary surfactant. It is well established that pulmonary surfactant plays a role of maintaining low surface tension at the air-liquid interface of the alveoli. Consequently, one of the mechanisms of postischemic injury in deflated lung would be due to shear stresses during reexpansion by high surface tensions resulting from surfactant impairment. On the contrary, Steen and coworkers (19) demonstrated excellent lung function after 24-h storage in the deflated state in a porcine transplantation model. They hypothesized that lungs in a deflated state would reduce the core temperature more quickly and maintain pulmonary functions better than those in an inflated state. However, their hypothesis is weakened by the failure to compare the pulmonary functions of deflated lungs with those of inflated lungs or measure intrapulmonary temperature or high-energy phosphate during preservation.

Because the mechanisms of lung injury caused by anaerobic or deflated conditions have not been fully clarified, there has been some confusion. The results of our present study indicate that, in order to determine the optimal state of the alveoli during lung storage, the O₂ content and alveolar inflation should be analyzed separately. The present study did not investigate the optimal quantity of oxygen or alveolar inflation. However, the energy concentration was maintained sufficiently in the deflated lungs and there was no significant difference in the inflation pressure between the room air- inflated and N₂-inflated lungs after 6-h cold storage compared with the baseline values (88.3 ± 1.7%, 91.2 ± 1.5%, respectively). These results suggested that only a small quantity of oxygen will be required during cold storage. Therefore, in clinical lung storage, to suppress the production of oxygen radicals (21), it may be better for the lungs to be inflated with gases containing less oxygen. Further studies are necessary to clarify this hypothesis.

In conclusion, on cold storage of the lungs, anaerobic metabolism alone could not maintain the pulmonary energy concentration, and it tended to be associated with severer I-R injury. Although the energy level was maintained in the deflated lungs, deflation caused damage to the alveolar cells and alveolocapillary membranes which in turn induced mechanical injury owing to shear stresses during reexpansion. Consequently, inflation and the presence of oxygen during cold ischemia appear to be necessary for improving the postpreservation pulmonary function.