INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Lung transplantation has become an important modality for the treatment of end-stage lung disease. More than 1,000 lung-transplantation operations are now performed annually throughout the world (1). On the other hand, the shortage of lung donors, and ischemia-reperfusion (IR) injury following transplantation, have been grave problems in lung transplantation. To solve these problems, we have developed new organ-preservation solutions that can prevent tissue damage and extend ischemic periods with satisfactory results (2).

Nitric oxide (NO) has many physiologic activities, including a vasodilatory effect (6), inhibition of adhesion and migration of neutrophils to the vascular wall (7), and a protective action against cell damage caused by reactive oxygen species (ROS) (8). NO released from endothelial cells activates soluble guanylate cyclase in vascular smooth-muscle cells, with consequent production of cyclic guanosine-3',5'-monophosphate (cGMP), and plays an important role in maintaining the function of blood vessels (9). However, when endothelial cells are exposed to IR, endogenous NO reacts with superoxide, which is produced rapidly during reoxygenation, drastically reducing the available quantity of NO. Consequently, the tissue cGMP concentration falls, and vascular function deteriorates (10).

Inhaled NO (11), and NO donors such as N-nitro-L-arginine methyl ester and N-nitro-D-arginine methyl ester (12), attenuate IR injury. Nitroglycerin (NTG), another NO donor, improves lung preservation for transplantation (13). Because NTG has been used as a vasodilator through its release of NO for a prolonged period (14), it may be suitable for the supplementation of lung preservation solutions. Administration of NTG during the initial reperfusion of lung grafts has been reported to improve pulmonary function after reperfusion (15). However, the direct mechanisms by which NTG protects lung tissue against injury are unknown.

During IR, ROS are produced in massive amounts resulting in tissue damage. ROS also cause DNA damage and produce various oxidatively modified products (16). Of these products, 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most popular markers for the evaluation of oxidative damage to DNA (17). Although 8-OHdG has been measured with an electrochemical detector connected to a high-pressure liquid chromatography (HPLC) system, localization of oxidative DNA damage was not possible with this method, and artifacts generated during sample preparation have been a subject of debate (18). Subsequently, a monoclonal antibody (mAb) (N45.1) that has a high affinity for 8-OHdG was developed (19). In situ quantification of oxidative damage to DNA (8-OHdG Index) in tissues has become feasible through immunohistochemical staining with this antibody. Data obtained with this method correlate well with HPLC results. Furthermore, this method produces fewer artifacts than the HPLC method (17).

In the present study we investigated: (1) whether supplementation of an organ preservation solution with NTG could maintain the cGMP level in lung tissue during cold ischemia, and consequently improve postreperfusion pulmonary function; and (2) whether supplementation with NTG could attenuate oxidative DNA damage to reperfused lung, as evaluated with the 8-OHdG Index. An ex vivo perfusion circuit was used (20). With this model, we evaluated the hemodynamics and gas exchange of preserved lung during reperfusion, using mixed venous blood obtained from rats as a perfusate and the isolated lung as a biologic deoxygenator (3, 21).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Preservation Solution and Drugs

Extracellular-type Kyoto (ET-K) solution, which was used in the study, is an organ preservation solution with an ionic composition similar to that of extracellular fluid, and is supplemented with the nonreducing disaccharide trehalose, which has a cell membrane-protective effect (22). The composition of ET-K solution includes Na⁺, 100 mM; K⁺, 44 mM; gluconate, 100 mM; phosphate, 25 mM; trehalose, 4.1%, and hydroxyethyl starch, 3%. Its osmolarity is 366 mOsm/L, and its pH is 7.40. NTG was purchased from Green Cross, Ltd. (Osaka, Japan), and prostaglandin E1 (PGE₁) was kindly provided by Ono Pharmaceutical, Ltd. (Osaka, Japan).

Donor and Deoxygenator Lung Preparation

All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the National Institutes of Health (23). Surgical procedures were performed as described (3, 21), using inbred male Lewis rats (280 to 300 g; Seac Yoshitomi, Ltd., Fukuoka, Japan). Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg), and were intubated after tracheostomy. Ventilation conditions during surgery were maintained with room air at a tidal volume (VT) of 3.0 ml and a respiratory rate (RR) of 60 breaths/min, using a volume-controlled respirator (Model SN480-7; Shinano, Tokyo, Japan). After a median abdominal incision was made, donor rats were heparinized (400 units) via the inferior vena cava. A median sternotomy was performed and a 14-gauge pulmonary arterial cannula was inserted into right ventricular outflow tract. The abdominal aorta and vena cava, right and left ventricles, and left atrial appendage were incised to facilitate free flow of pulmonary venous blood. Positive end-expiratory pressure (PEEP) at 2 cm H₂O was then applied to the airway. For donor lung, the pulmonary vascular bed was flushed with the perfusate (4° C, 50 ml) containing PGE₁ (10 µg), at a pressure of 20 cm H₂O. After harvesting of the heart-lung block, airway pressure was kept at 14 cm H₂O, the trachea was clamped, and the right pulmonary hilus was ligated. The right upper and middle lobes were resected and frozen for measurement of tissue cGMP level. The heart-lung block was then immersed in the preservation solution at 4° C.

For deoxygenator lungs, harvesting was done as with the donor lungs, but without the flushing procedure. Both sides of lungs were used as deoxygenators.

Perfusion Circuit

We used an ex vivo rat lung perfusion model first described by DeCampos and colleagues (Figure 1) (20). The perfusion circuit was filled with 40 ml of fresh heparinized blood taken from three rats. The blood was kept at 37° C. Right lower and mediastinal lobes of the lungs used for experimentation were resected for measurement of tissue cGMP level immediately after cold preservation for 15 h. The left lung was placed in a chamber maintained at 37° C with a humidity of 100%. The left lung was ventilated at an inspired oxygen fraction (FI_O2) of 1.0, VT of 1.5 ml, and RR of 40 breaths/min, with a PEEP of 2 cm H₂O. The deoxygenator lung was maintained under the same conditions and ventilated with mixed gas (FI_O2 = 0.04, inspired carbon dioxide fraction = 0.08, and inspired nitrogen fraction = 0.88) at a VT of 3.0 ml and a RR of 60 breaths/min, with a PEEP of 2 cm H₂O. The blood deoxygenated by the deoxygenator lung was perfused through the experimental lung for oxygenation and was then recirculated to the deoxygenator lung with a roller pump (Model 7553-80; Cole Parmer Instrument Co., Chicago, IL). The flow rate of perfusion was gradually increased to 4 ml/min over the initial 10 min, and was maintained at 4 ml/min thereafter, until the end of the study. The perfused blood was adjusted with sodium bicarbonate if necessary to keep the pH of blood returning from the deoxygenator lung between 7.35 and 7.45. 

View larger version (100K): [in this window] [in a new window]   Figure 1.   Schematic illustration of a perfusion circuit. The single left lobe of the experimental lung was ventilated with 100% oxygen and the deoxygenator lung was ventilated with a hypoxic mixture (4% oxygen, 8% carbon dioxide, and 88% nitrogen). The circuit was filled with 40 ml of heparinized blood pooled from three rats. The blood from the deoxygenator lung was perfused through the experimental lung for oxygenation, and was then recirculated to the deoxygenator lung. This model was set up in a chamber maintained at 37° C and 100% humidity.

When severe pulmonary edema of the experimental lung occurred and fluid exuded from the tracheal tube, reperfusion was discontinued.

Experimental Groups

Rats were allocated to each of three groups at random. In Fresh group (n = 7), lungs were flushed only with ET-K solution and were immediately reperfused for 60 min. In the groups designated NTG+ (n = 10) and NTG (n = 10), lungs were flushed with the perfusate with and without NTG (0.44 mM), respectively, before being immersed in the corresponding solution for 15 h at 4° C and reperfused for 60 min.

Physiologic Measurements

Pulmonary venous blood from the lung used for experimentation or from the deoxygenator lung, coming from the incised left atrium and ventricle, was collected for blood gas analysis. The analyses were performed immediately before reperfusion and then every 10 min during reperfusion, with an automatic blood gas analyzer (ABL300; Radiometer A/S, Copenhagen, Denmark). The data were used to calculate the pulmonary shunt fraction (S/T) with the following formula:

(1)

where Cc, Ca, and Cv represent the oxygen content of pulmonary capillary, pulmonary arterial, and pulmonary venous blood, respectively. Mean pulmonary arterial pressure () and peak inspiratory airway pressure (Ppi) were monitored continuously and recorded at 10-min intervals with a pressure-monitoring system (AP-641G; Nihon Kohden, Tokyo, Japan). The lower one-third of the left lung was resected at the end of reperfusion, and the wet tissue was weighed. The tissue was then dried at 55° C for 72 h, the dry tissue was weighed, and the wet-to-dry weight ratio of the lung tissue (W/D ratio) was calculated. For assessment of stability of the ex vivo perfusion system, the W/D ratio of the left lung tissue of the deoxygenator lung was evaluated similarly.

cGMP Assay

For cGMP assays, right upper and middle lobes excised immediately after flushing, right lower and mediastinal lobes excised before reperfusion, and one-third of the left lung excised after reperfusion were snap frozen in liquid nitrogen and kept until the time of assay for tissue cGMP level. The lung tissue samples used for assay were homogenized in ice-cold 0.1 N HCl (2 ml). After centrifuging the homogenates at 3,000 × g for 15 min at 4° C, we measured cGMP in the supernatants with a radioimmunoassay kit (cGMP Assay kit; Yamasa, Chiba, Japan). Results were reported as picomoles of cGMP per milligram of protein. The protein content of samples was quantified according to the method of Lowry and coworkers (24).

Myeloperoxidase Assay

A myeloperoxidase (MPO) assay was done on the experimental lungs that were resected 60 min after reperfusion and snap frozen in liquid nitrogen until the assay. Tissue was homogenized in 2 ml of potassium phosphate buffer (50 mM, pH 6) containing hexadecyltrimethylammonium bromide (0.5%; Sigma Chemical Co., St. Louis, MO). The sample was centrifuged at 12,000 × g for 10 min, and the supernatant was decanted. The MPO activity in each sample was measured with a standard chromogenic spectrophotometric technique (25).

Immunohistochemistry with Monoclonal Antibody N45.1

Sample tissues from the lungs for experimentation were fixed overnight with Bouin's solution after reperfusion for 60 min. In addition, normal lung tissues from three rats that were anesthetized and ventilated with room air were fixed at identical settings immediately after excision, so that the lungs had neither been subjected to flushing nor to preservation or reperfusion. The avidin-biotin complex method (26), with a slight modification, was used for immunohistochemical staining. After deparaffinization of lung tissue with xylene and ethanol, normal rabbit serum (diluted to 1:75; Dako, Kyoto, Japan) was used to block nonspecific binding, and mAb N45.1 (0.5 µg/ml), biotin-labeled rabbit antimouse IgG serum (diluted to 1:300; Dako), and avidin-biotin-alkaline phosphatase complex (diluted to 1:100; Dako) were sequentially applied. The final color development reaction was run simultaneously for 24 specimens (nine specimens from the NTG+ group, eight from the NTG group, four from the Fresh group, and three from normal lungs).

To confirm the specificity of immunostaining, we conducted the 8-OHdG absorption tests by incubating an excess of 8-OHdG (final concentration, 500 µM; Wako, Osaka, Japan) with the primary antibody on glass slides. Alternatively, to produce a negative control, we omitted the first antibody from the staining procedure.

Quantification of Immunohistologic Data

Quantification of immunohistologic data was done as previously described, with the 8-OHdG Index (17). For this purpose, specimens for 8-OHdG immunohistochemistry were used for densitometric analysis. Color slides (35 mm) of three appropriate locations, which were focused on bronchial epithelial cells (Br-EC), alveolar epithelial lining cells (Al-EC), or pulmonary arterial endothelial cells (Pa-EC), were taken of each specimen at a magnification of 40 × 5, covering an area of approximately 335 µm × 220 µm. Color images were obtained as PICT files with a slide scanner (Quick Scan; Minolta, Tokyo, Japan) connected to a Power Macintosh 8500/1200 computer (Apple Computer Japan, Inc., Tokyo, Japan). The brightness and contrast of each image file were uniformly enhanced with Adobe Photoshop version 3.0J, followed by image analysis done with NIH Image freeware (version 1.61; National Institutes of Health, Bethesda, MD; available on the internet via a file transfer protocol from zippy.nimh. nih.gov). The following equation was used for quantification of immunohistochemical data:

(2)

where X is staining density, indicated by a number between 0 and 256 on the Gray scale. Specimens in the three groups were analyzed for Br-EC and Al-EC within a particular respiratory area, and for Pa-EC within a particular vessel area, respectively. The mean of the data obtained from three independent files was used as a representative value for each animal.

Statistical Analysis

All results are presented as mean ± SEM. Comparison of groups with more than two representatives was done through one-way or two-way analysis of variance (ANOVA), followed by the Student-Newman- Keuls test (27), with significance defined at p < 0.05. Comparison of two groups was done with Student's t test. These analyses were done with an IBM PC computer (IBM Japan, Ltd., Tokyo), using SigmaStat for Windows, version 1.0 (Jandel Corporation, San Rafael, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

All lungs from animals in the Fresh and NTG+ groups were reperfused throughout the entire 60-min observation period. However, ventilation of one lung from the NTG group (n = 10) was discontinued after 50 min of reperfusion because of severe pulmonary edema.

The Stability of the Ex Vivo Perfusion System

Hemoglobin levels of the priming blood in the perfusion circuit were 13.7 ± 0.7 mg/dl in the Fresh group, 14.3 ± 0.3 mg/dl in the NTG+ group, and 14.3 ± 0.2 mg/dl in the NTG group, with no significant differences among the three groups. There was no significant difference in the blood gas analysis data for blood from the deoxygenator lungs of the three study groups before and throughout the reperfusion perfusion (Table 1). The W/D ratios of the deoxygenator lungs after reperfusion were 6.5 ± 1.3 in the Fresh group, 6.9 ± 1.0 in the NTG+ group, and 6.1 ± 0.4 in the NTG group, with no significant differences among the three groups. Thus, the ex vivo perfusion system was very stable through the experimental period.

Lungs Perfused without NTG Showed Features of IR Injury

In the Fresh group, lungs reperfused immediately after harvesting, without preservation, showed good pulmonary function. The W/D ratio for these lungs was 6.2 ± 0.6 (Figure 2), resembling that of normal lungs, as we previously reported (3, 21). Shunt fractions remained at 4% to 6% (Figure 3). and Ppi values were within 15 to 16 mm Hg and 11 to 12 mm Hg of one another, respectively (Figure 4), during the 60-min reperfusion period. In the absence of NTG, reperfusion induced severe pulmonary edema and deterioration of pulmonary function. W/D ratios in the NTG group increased by about threefold over those of the Fresh group, indicating severe pulmonary edema (Figure 2). During the reperfusion, the shunt fractions gradually increased by up to 40%, and the Ppi values were over 20 mm Hg (Figures 3 and 4B). On the other hand, values did not change very greatly throughout the reperfusion period (Figure 4A).

View larger version (26K): [in this window] [in a new window]   Figure 2.   Lung W/D ratio after reperfusion. Single-left lungs were reperfused after hypothermic preservation for 15 h in ET-K solution alone (NTG group, n = 9) or in ET-K solution plus nitroglycerin (NTG+ group, n = 10), or were immediately reperfused after being freshly flushed with ET-K solution alone (Fresh group, n = 7). The lung W/D ratio after reperfusion for 60 min in the NTG group was significantly greater than in the Fresh group, which was inhibited by NTG treatment (p < 0.01, one-way ANOVA).* p < 0.05 versus Fresh group; ** p < 0.05 versus NTG group, Student-Newman-Keuls test. Values are mean ± SEM.

View larger version (18K): [in this window] [in a new window]   Figure 3.   Shunt fraction of experimental lungs during reperfusion. The shunt fraction of lungs of the NTG group (n = 10) was greater than those of the other two groups (p < 0.001, two-way ANOVA). * p < 0.05 versus NTG+ (n = 10) and Fresh (n = 7) groups; ** p < 0.05 versus Fresh group, Student-Newman-Keuls test. Values are mean ± SEM.

View larger version (18K): [in this window] [in a new window]   Figure 4.   Physiologic assessments of experimental lungs during reperfusion. (A) Ppa. There were no significant differences among the three study groups as analyzed with two-way ANOVA. (B) Ppi. The Ppi of the NTG group was significantly greater than those of the other two groups (p < 0.05, two-way ANOVA). The Ppi of the NTG+ group was significantly greater than that of the Fresh group (p < 0.05, two-way ANOVA). * p < 0.05 versus Fresh group, Student-Newman-Keuls test. Values are mean ± SEM.

Supplementation of the Perfusate with NTG Improved Pulmonary Function after Reperfusion

In the NTG+ group, NTG was added to both the flush and the preservation solutions. A significant improvement in gas exchange was observed over that in the NTG group. Shunt fractions were significantly lower than those in NTG group until 50 min after reperfusion (Figure 3). There were no significant differences in shunt fractions between the NTG+ and Fresh groups. W/D ratios were significantly lower than those in the NTG group (p < 0.05) (Figure 2). Interestingly, was unaffected by NTG treatment (Figure 4A), whereas values of Ppi were significantly lower than those in NTG group throughout the experiment (Figure 4B).

Insufficient Inhibitory Effect of NTG against Neutrophil Infiltration

MPO activity has been commonly used as a marker for neutrophil infiltration and activation during acute inflammatory responses. We found no significant difference in MPO activity between the NTG+ group (254.9 ± 41.0 U/mg protein) and the NTG group (295.8 ± 51.2 U/mg protein). The MPO assay was not done in the Fresh group.

Changes In Tissue cGMP Level during Preservation and Reperfusion

Levels of cGMP in lung tissue declined during preservation and reperfusion. In the NTG group, tissue cGMP levels decreased to about one-third of those immediately after flushing following preservation of tissue at 4° C for 15 h, and to less than one-ninth of the levels after flushing following reperfusion for 60 min (Figure 5). NTG added to the perfusate maintained tissue cGMP levels after preservation at significantly higher values than those in the NTG group (p < 0.05), but did not maintain cGMP levels during reperfusion (Figure 5).

View larger version (26K): [in this window] [in a new window]   Figure 5.   Levels of cGMP in lung tissue. Tissue cGMP levels were measured with a radioimmunoassay after flushing of lungs with ET-K solution alone (Fresh and NTG groups) or with ET-K solution plus nitroglycerin (NTG+ group), following preservation for 15 h at 4° C in the NTG and NTG+ groups, and after reperfusion for 60 min (n = 4 for the Fresh group, n = 10 for the NTG+ group, and n = 9 for the NTG group). Values are mean ± SEM. There were no significant differences among the three groups after flushing and reperfusion, as analyzed with one-way ANOVA. After storage, cGMP was significantly increased in the NTG+ group as compared with the NTG group. * p < 0.05 versus NTG group, Student's t test.

Immunohistochemical Detection of Sites of Oxidative Damage Caused by IR Injury

In normal lung, nuclear staining with mAb N45.1 was barely detectable. Reperfusion without hypothermic preservation slightly increased the staining. Oxidative damage caused by IR injury was found in both airway epithelium and blood vessel endothelium. In the NTG group, intense nuclear staining of bronchial epithelial cells, alveolar epithelial cells, and pulmonary endothelial cells was observed. NTG treatment significantly inhibited the IR-induced oxidative damage seen in the NTG group (Figures 6-8). No staining was observed either when the N45.1 antibody was omitted or when the primary antibody applied to the slides was preabsorbed with an excess of 8-OHdG (data not shown).

View larger version (87K): [in this window] [in a new window]   Figure 6.   Immunohistochemical staining for 8-OHdG in bronchial epithelial cells of lungs after reperfusion. Immunostaining of specimens of lungs in from the Fresh (B), NTG (C ), and NTG+ (D) groups after reperfusion (original magnification ×200). In addition, normal lung tissues collected from rats that were anesthetized and ventilated with room air were fixed under identical conditions immediately after excision (A). The normal lungs had not been subjected to flushing, preservation, or reperfusion. All specimens were examined with mAb N45.1 for 8-OHdG. The nuclear staining in normal lungs was very weak. Intense nuclear staining of Br-EC was observed in the NTG group, whereas faint nuclear staining was seen in the Fresh and NTG+ groups. Bar = 20 µm.

View larger version (78K): [in this window] [in a new window]   Figure 7.   Immunohistochemical staining for 8-OHdG in alveolar epithelial lining cells of lungs after reperfusion. Immunostaining of specimens of normal lungs (A), and of lungs following reperfusion from the Fresh (B), NTG (C), and NTG+ (D) groups (original magnification ×200). All specimens were examined with mAb N45.1 for 8-OHdG. Nuclei of Al-EC in the NTG group were stained most intensely, and those in the NTG+ and Fresh groups were less intensely stained. Faint nuclear staining was seen in normal lungs. Bar = 20 µm.

View larger version (87K): [in this window] [in a new window]   Figure 8.   Immunohistochemical staining for 8-OHdG in pulmonary arterial endothelial cells of lungs following reperfusion. Immunostaining of specimens of normal lungs (A), and lungs following reperfusion from the Fresh (B), NTG (C), and NTG+ (D) groups (original magnification ×200). All specimens were examined with mAb N45.1 for 8-OHdG. Intense nuclear staining of Pa-EC was observed in the NTG group, whereas faint staining was seen in the NTG+ and Fresh groups. Faint nuclear staining was seen in normal lungs. Bar = 20 µm.

Quantification of Oxidative DNA Damage in Postreperfusion Lung

Oxidative DNA damage in Al-EC was much more severe than that to the other two types of cells examined. In the NTG group, 8-OHdG Indices of Al-EC were about fivefold greater than those of Br-EC (p < 0.05) when quantified by morphometric analysis (Figure 9). Supplementation of the perfusate with NTG attenuated oxidative stress caused by IR injury. 8-OHdG Indices of the NTG group were 12 times greater in Al-EC, 19 times greater in Pa-EC, and six times greater in Br-EC, respectively, than those of the NTG+ group. In the Fresh group, 8-OHdG indices for all three areas were below 20, and were significantly below those in the NTG group (Figure 9).

View larger version (26K): [in this window] [in a new window]   Figure 9.   8-OHdG Index of lungs following reperfusion. The 8-OHdG Index was calcuated through immunostaining of specimens with mAb N45.1 for Br-EC, Al-EC, and Pa-EC (n = 9 for each cell type), in the Fresh, NTG and NTG+ groups. Values are mean ± SEM. 8-OHdG Indices of the NTG group were significantly higher than those of the Fresh and NTG+ groups for each site examined (Br-EC p < 0.01; Al-EC: p < 0.001; and Pa-EC: p < 0.0001, one-way ANOVA). * p < 0.05 versus Fresh group; and ** p < 0.05 versus NTG group, Student-Newman-Keuls test). The 8-OHdG Index of the NTG group for Al-EC was significantly higher than that for Br-EC (p < 0.05, one-way ANOVA). # p < 0.05 versus NTG group for Br-EC, Student-Newman-Keuls test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Endogenously produced NO stimulates production of cGMP, which acts as an intracellular second messenger in relaxing vascular smooth muscle (9), inhibiting platelet adhesion and aggregation (28), and adjusting vascular permeability (29). Levels of endogenous NO produced in the lung and heart have been reported to be decreased during preservation and reperfusion, through a response to ROS generated from alveolar macrophages and neutrophils (10). NTG is known to work in vivo as an NO donor. Intravenous infusion of NTG into rabbits (30, 31) or lambs (32) induced dose-dependent increments in exhaled NO and concomitant reductions in systemic blood pressure. Marczin and colleagues showed that intravenous administration of NTG resulted in an increase in exhaled NO that coincided with a reduction of arterial blood pressure in patients undergoing open heart surgery (33). In the present study, cGMP levels in lung tissues decreased during preservation in the NTG group. Supplementation of the perfusate with NTG maintained tissue cGMP levels during cold preservation, indicating that NTG may play a role as an NO donor during lung preservation for 15 h. David and coworkers reported that the effect of NTG could persist for 18 to 24 h (14). The tissue cGMP level in the NTG+ group diminished after reperfusion. The volume of the vascular bed of the rat left lung is estimated to be approximately 1 ml, and the total blood volume in the perfusion circuit was about 40 ml. Preservation solution in the vascular bed is promptly washed out at the beginning of reperfusion. Consequently, the amount of NTG within the pulmonary vascular bed is quite small. Furthermore, the production of NO seen in normal lung may be inhibited by ROS produced by IR. Hogg and coworkers demonstrated that endogenous NO reacts directly with superoxide at the endothelial surface during reperfusion, to produce hydroxyl radical (34). Therefore, the higher tissue cGMP level in the NTG+ group in our study could not be maintained during reperfusion.

Nevertheless, addition of NTG to the preservation solution protected lungs against severe IR-induced pulmonary edema and dysfunction. The shunt fraction and Ppi in the NTG group gradually increased over the course of reperfusion, and the W/D ratio in this group was significantly higher than that in the Fresh group. Addition of NTG improved shunt fractions throughout reperfusion, which were significantly better than the fractions in the NTG group. By contrast, NTG had no effect on or MPO activity in our study. These results suggest that the protective effects of NTG in preservation solution may be not related to vasodilation or inhibition of neutrophil adhesion or activation.

NTG may function via a cGMP-independent mechanism (35). Zhou and associates showed that relaxation of canine trachea induced by the NO donor sodium nitroprusside was potentiated by methylene blue and hemoglobin, which reduced cGMP accumulation (34). Another NO donor, S-nitrosoglutathione, was also reported to relax canine tracheal smooth muscle, in part through a cGMP-independent process (36). It was recently reported that NO could induce the synthesis of glutathione, an important intracellular antioxidant, through a cGMP-independent pathway (37).

Our results demonstrate a strong correlation between the inhibition of oxidative DNA damage as quantified with the 8-OHdG Index and improved postreperfusion pulmonary function in our NTG+ group. 8-OHdG Indices in the Fresh group were very low, and those in the NTG group were significantly increased in all three cell areas that we examined. NTG treatment blocked the increase in the 8-OHdG index, keeping it at to the levels seen in the Fresh group. NTG has been reported to have a cytoprotective effect on H₂O₂-induced endothelial dysfunction in cell culture (38), to improve rat lung preservation through antineutrophil and antiplatelet (13), and to reduce plasma malondialdehyde levels after thrombolytic therapy for acute myocardial infarction (39). The cytoprotective effect of NTG seen in the present in study may contribute significantly to the amelioration of IR injury of the lung. NO has been suggested to have a protective effect against damage caused by ROS (8). Inhaled NO inhibits capillary damage caused by oxygen radicals (40).

8-OHdG Indices for the NTG group in our study were in the order Al-EC > Pa-EC > Br-EC; accordingly, oxidative damage to DNA in alveoli was the most severe among the three areas that we examined. This result may be attributable to the following events: (1) During preservation, endothelia in the vascular bed are in direct contact with the preservation solution, which may have cell membrane-protective effects. (2) After reperfusion, the capillary endothelial cells lining alveoli are exposed to reperfusion flow. Damage to endothelial cells may consequently affect alveolar epithelial cell function. In addition, alveolar epithelial cells are exposed to a high concentration of oxygen, and alveoli can therefore also be damaged directly through the respiratory tract. (3) When exposed to oxygen, alveolar epithelial cells are more readily affected by oxidative stress than are bronchial epithelial cells, because type I alveolar epithelial cells are flatter and have a larger surface area than bronchial epithelial cells (41). (4) The respiratory tract lining fluid contains antioxidants such as mucin, uric acid, and reduced glutathione, and the thickness of the lining fluid layer decreases from bronchi to alveoli (41). In this study we focused on pulmonary arterial endothelium to evaluate DNA damage. Recently, Khimenko and Taylor reported that the greatest damage occurred within postalveolar venules in isolated perfused rat lung after IR (44). Prudent observation of small vessels will further elucidate the predominant site of injury from IR.

In summary, a cytoprotective effect of NTG given during flushing and preservation of lung was observed in alveolar and bronchial epithelial cells, as well as in pulmonary arterial endothelial cells, after reperfusion. This cytoprotective effect was associated with and may contribute to amelioration of IR-induced lung injury. NTG could be a potent additive to preservation solutions for attenuating DNA damage caused by oxidative stress, thereby protecting against reperfusion injury during lung transplantation.