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Ann Thorac Surg 1996;62:1644-1649
© 1996 The Society of Thoracic Surgeons

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Original Articles: General Thoracic

Contralateral Lung Injury Associated With Single-Lung Ischemia-Reperfusion Injury

Second Department of Surgery and Third Department of Medicine, Sapporo Medical University and Hospital, Sapparo, Japan

Accepted for publication July 22, 1996.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Background. There have been very few studies on the effect of single-lung ischemia-reperfusion on the function of the contralateral lung. This study was designed to clarify the effect.

Methods. Fifteen mongrel dogs were divided into two groups. In group 1 (n = 7), the left lung was subjected to ischemia without ventilation for 90 minutes, and then reperfused. In group II (n = 8), the lung was not subjected to ischemia, and was ventilated during the 90-minute ischemia of group I. Arterial blood gas, hemodynamics, extravascular lung water, and airway pressure were measured. Pulmonary biopsy was performed to evaluate adenine nucleotide levels. The protein concentration and phosphorous concentration of phospholipids in bronchoalveolar lavage fluid were measured.

Results. Group I, with perfusion and ventilation of the right lung alone, was significantly inferior to group II with respect to arterial blood gas, right pulmonary compliance, extravascular lung water of the right lung, and the protein concentration in the bronchoalveolar lavage fluid of the right lung after the 90-minute period.

Conclusions. These results indicate that 90 minutes of warm ischemia and reperfusion of the left lung caused deterioration of not only the left but also contralateral right pulmonary function.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Ischemia and reperfusion is an unavoidable process during transplantation. Cells that are subjected to ischemia and reperfusion are injured. Toxic oxygen metabolites in the form of superoxide anions and hydrogen peroxides [1] are generated, and phospholipase is activated after ischemia and reperfusion. Phospholipase activation causes the cellular membrane to break down, and arachidonic acid is released. Once arachidonic acid is released, the formation of arachidonic acid metabolites derived from the cyclooxygenase pathway occurs, which has been associated with ischemia-reperfusion injury [2, 3], and the secretion of specific chemical mediators is promoted. The mediators can activate the complement and coagulation systems, resulting in the release of a potent soluble mediator and an inflammatory response. Although the process following ischemia and reperfusion has been generally studied only in the relevant cells and organs, it is thought that the mediators cause injury to cells and organs without ischemia, if they reach the cells and organs. The purpose of the present study is to investigate this "ischemia-reperfusion–related injury" using a canine hilar stripped lung model with additional 90-minute warm ischemia.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Surgical Manipulations
Fifteen mongrel dogs were randomly divided into two groups. After left hilar stripping, in group I (n = 7), the left lung was subjected to ischemia without ventilation and inflation for 90 minutes and then reperfused. In group II (n = 8), the lung was not subjected to ischemia and was ventilated during the 90-minute ischemia of group I.

The dogs were anesthetized with thiopental sodium (20 mg/kg) and intubated after one venous line was established. Anesthesia was maintained with thiopental sodium (20 mg•kg^-1•h^-1), and the venous line was maintained with Ringer's lactate.

The dogs were placed in the supine position and ventilated during the experiments with 100% oxygen at a rate of 12 to 20 cycles/min, tidal volume of 20 mL/kg, and positive end-expiratory pressure of 5 cm H₂O. A Swan-Ganz catheter was positioned into the main pulmonary artery through the jugular vein. A systemic artery pressure line (7F introducer) was positioned into the femoral artery, and an extravascular lung water catheter (Surgitherm balloonless catheter; Electron Catheter Co, Rahway, NJ) was positioned into the abdominal aorta through the introducer. An electrocardiogram, systemic artery pressure, pulmonary artery pressure, right atrial pressure, and airway pressure were continuously monitored. After median sternotomy and left-sided pericardiectomy, both the main pulmonary arteries, the left pulmonary veins, and both the main bronchi were isolated, and tape ligatures were passed around them. Left bronchial arteries and the bronchial rami of the vagus nerve were cut. A left atrial pressure line was placed into the left atrium through the left atrial appendage. Heparin (100 U/kg) was administered systemically.

Physiologic Measurements
A recovery period from the operative damage of approximately 1 hour was allowed for arterial oxygen tension (PaO₂) to recover to the preoperative value. Dogs were excluded from the experiments if the PaO₂ did not recover to the preoperative value during this period. After a 5-minute occlusion of the right main pulmonary artery and main bronchus, baseline functional data of the left single lung, which had been ventilated and perfused, were obtained from all dogs. These included systemic arterial, pulmonary arterial, both atrial, and airway pressures. Arterial blood gases were analyzed using an ABL-30 acid base analyzer (Radiometer A/S, Copenhagen, Denmark). Cardiac output was determined in triplicate by the thermodilution method. Pulmonary arterial resistance was calculated by the standard formula. Extravascular lung water volume was determined on the basis of the thermal-sodium double indicator dilution method using a 3% cold saline solution (1 to 3 mL), which was injected into the right atrium (ETV1100; Nihon Kohden Co, Tokyo, Japan). Pulmonary compliance was calculated using the following formula: pulmonary compliance = tidal volumes/peak airway pressures with zero end-expiratory pressure. After data sampling with perfusion and ventilation of the left lung alone, the right main pulmonary artery and main bronchus were released. Baseline functional data with perfusion and ventilation of the right lung alone were obtained in the same manner as data sampling of the left single lung after 5-minute occlusion of the left main pulmonary artery and main bronchus. After baseline data sampling of both the lungs in group I, the left pulmonary artery, the left main bronchus, and pulmonary veins were occluded without inflation, the left main pulmonary artery, veins, and bronchus were released, and the left lung was reperfused and ventilated after 90 minutes of warm ischemia. In contrast, the left lung was continuously perfused and ventilated in group II during the 90-minute warm ischemia in group I. The functional data in group II were obtained at the same point and in the same manner as in group I.

Assessment of Bronchoalveolar Lavage Fluid
After all the functional data sampling, at 210 minutes after the 90-minute ischemia in group I or nonischemia in group II, 100 mL of normal saline solution was directly instilled into the bilateral lower lobes using a Foley balloon catheter (8F; C. R. Bard, Inc, Billerica, MA). Bronchoalveolar lavage fluid (BALF) of both the lower lobes was obtained and pooled to yield a single specimen. The aspirated BALF was centrifuged at 150 g for 10 minutes. The resulting supernatant was recovered and stored at -20°C, or immediately used for analysis as described below. The total nonsedimentable protein in the BALF was measured by a modified Lowry method [4]. The organized phosopholipid was extracted and purified by the method described by Bligh and Dyer [5], and the phosphorus of organized phospholipid was measured as described by Bartlett [6].

Assessment of Adenine Nucleotide Levels in Lung Tissue
At 210 minutes after the 90-minute ischemia in group I or nonischemia in group II, a lung biopsy of the bilateral middle lobes was carried out and the biopsy samples were immediately frozen and stored in liquid nitrogen. Adenine nucleotides in each biopsy specimen were extracted with 0.5 N perchloric acid (4°C) and neutralized. The levels of cellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were determined using high-performance liquid chromatography (HPLC system; Nihon Bunko Co Ltd, Tokyo, Japan) after the 90-minute period. Energy charge (EC) was calculated by the following formula: EC = (ATP + 0.5 x ADP)/(ATP + ADP + AMP).

Statistics
All values were expressed as mean ± standard deviation of the mean. Paired and independent sample comparisons were made for all experimental results by means of paired and unpaired Student's t tests. Statistical significance was accepted at a 95% confidence limit (p < 0.05).

Animal Care
All dogs 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 Sciences and published by the National Institutes of Health (NIH publication 85-23, revised 1985).

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Arterial Blood Gas Analysis and Hemodynamics
The baseline data of arterial blood gas analysis and hemodynamics were not significantly different between the two groups. The PaO₂ with perfusion and ventilation of the left lung alone was significantly less in group I than in group II at 2 to 3 hours after the 90-minute period: 427 ± 150 mm Hg versus 595 ± 25 mm Hg at 2 hours after the 90-minute period (p < 0.02) and 360 ± 220 mm Hg versus 572 ± 41 mm Hg at 3 hours after the 90-minute period (p < 0.03), respectively. The PaO₂ with perfusion and ventilation of the right lung alone was significantly less in group I than in group II at 1 to 3 hours after the 90-minute period: 508 ± 75 mm Hg versus 589 ± 37 mm Hg at 1 hour after the 90-minute period (p < 0.03) and 362 ± 219 mm Hg versus 577 ± 37 mm Hg at 3 hours after the 90-minute period (p < 0.05). The PaO₂ of group I or group II with perfusion and ventilation of the left lung alone was similar to that obtained with perfusion and ventilation of the right lung alone throughout the experiments. The alveolar-arterial oxygen pressure difference (P(A-a)O₂) with perfusion and ventilation of the left lung alone was significantly greater in group I than in group II at 2 to 3 hours after the 90-minute period: 241 ± 145 mm Hg versus 83 ± 28 mm Hg at 2 hours (p < 0.02) and 302 ± 207 mm Hg versus 104 ± 40 mm Hg at 3 hours (p < 0.03), respectively. The P(A-a)O₂ with perfusion and ventilation of the right lung alone was significantly greater in group I than in group II at 1 to 3 hours after the 90-minute period: 166 ± 73 mm Hg versus 90 ± 34 mm Hg at 1 hour (p < 0.05) and 315 ± 221 mm Hg versus 106 ± 65 mm Hg at 3 hours (p < 0.05). The P(A-a)O₂ with perfusion and ventilation of the right lung was significantly greater at 2 hours after the 90-minute period in group II than that with perfusion and ventilation of the left lung alone. Cardiac output and pulmonary arterial resistance with perfusion and ventilation of the right or left lung alone after the 90-minute period in group II were not significantly different from those in group I. Pulmonary arterial resistance with perfusion and ventilation of the right lung alone was significantly less at baseline and 1 hour after the 90-minute period in group I than that with perfusion and ventilation of the left lung alone. Pulmonary arterial resistance with perfusion and ventilation of the right lung alone was significantly less in group II throughout the experiments than that with perfusion and ventilation of the left lung alone (Table 1).

Extravascular Lung Water Volume
There were no significant differences in the baseline extravascular lung water with perfusion and ventilation of the left lung alone between the two groups. Extravascular lung water of the left single lung and the right single lung at 3 hours after the 90-minute period were significantly greater in group I than in group II: 11.63 ± 5.63 mL/kg versus 7.14 ± 1.18 mL/kg for the right lung (p < 0.05) and 9.67 ± 3.71 mL/kg versus 6.06 ± 1.55 mL/kg for the left lung (p < 0.05). There were no significant differences in EVLW of group I or group II between the perfused and ventilated single lungs (see Table 1).

Protein Concentration and Phosphorus Concentration of Organized Phospholipid in Bronchoalveolar Lavage Fluid
The protein concentration in BALF of the left lower lobe was significantly greater at 210 minutes after the 90-minute period in group I than in group II: 262 ± 150 mg/mL versus 115 ± 52 mg/mL (p < 0.03). The protein concentration in BALF of the right lower lobe was also greater in group I than in group II: 192 ± 132 mg/mL versus 113 ± 35 mg/mL (p = 0.152). There was no significant difference in the protein concentration of BALF in group I or group II between the right and left lower lobes.

The phosphorus concentration of organized phospholipid in BALF of left and right lower lobes showed no significant difference between the two groups. There was also no significant difference in the phosphorus concentration of organized phospholipid of BALF in group I or group II between the right and left lower lobes (Table 2).

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment References

The lung is a unique organ, as it has two blood supplies: pulmonary arteries with low oxygen tension and bronchial arteries with high oxygen tension. The latter blood supply to the left lung disappears in this model as it does in clinical lung transplantation. The blood flow from the pulmonary artery is almost equal to the cardiac output, and therefore the right lung is exposed to a blood flow as high as the cardiac output during warm ischemia of the left lung in group I. The blood flow remains high immediately after reperfusion because of the no-reflow phenomenon of the left lung caused by hypoxic pulmonary vasoconstriction and microembolism, and this high pulmonary blood flow may cause mechanical impairment of pulmonary endothelial cells. Extravascular lung water volume may increase because of high blood flow. Some of the free radicals, proteases, and the other mediators produced by the warm ischemia-reperfusion injury are washed out and released into the blood stream, and the mediators that remain active without being metabolized must reach the contralateral right lung and injure it. The impairment induced by mediators released into the bloodstream may be larger in the contralateral right lung than in the left lung with warm ischemia, because the contralateral right lung has a higher blood flow than the left lung with pulmonary vasoconstriction and microembolism after ischemia.

There have been very few studies on the effect of ischemia-reperfusion after single-lung transplantation on the function of the contralateral lung. This study was designed to answer the following questions: (1) Does the ischemia-reperfusion of the left lung affect the nonischemic right lung? (2) If so, what is the effect? (3) What is the time-course of the effect?

Although we used a canine hilar stripped lung model with 90 minutes of warm ischemia instead of an autotransplantation model, we believe that the purpose of this study concerning ischemia-reperfusion injury was achieved using this model. The model is very simple, and the operative damage is little.

Arterial oxygen tension and P(A-a)O₂ with perfusion and ventilation of nonischemic right lung alone were significantly worse at 1 to 3 hours after the 90-minute period in group I than in group II. Extravascular lung water with perfusion and ventilation of the nonischemic right lung alone was significantly greater at 3 hours after the 90-minute period in group I than in group II. The pulmonary compliances of the left lung and the right lung were significantly less at 2 to 3 hours after the 90-minute period in group I than in group II. These results indicate that a 90-minute period of warm ischemia and reperfusion of the left lung contribute to impairment of not only the ipsilateral but also the contralateral right pulmonary function. The degree of impairment in the nonischemic lung is as great as that in the ischemic lung in group I. This is the reason why the ischemic lung is influenced by mild operative damage, reperfusion injury, and ischemia, while the nonischemic contralateral lung is affected by little operative damage and more severe injury caused by mediators that are produced by ischemia-reperfusion injury of the left lung. The pulmonary arterial resistance of the ventilated and perfused right lung was less at baseline to 3 hours after the 90-minute period in group II than that of the ventilated and perfused left lung. This difference depends on the difference in the degree of operative damage between the right and left lungs: mechanical injury caused by operative manipulation of the lung induces an increase in pulmonary arterial resistance. The pulmonary compliance in group I was greater at 1 hour after reperfusion in the perfused and ventilated right lung than in the perfused and ventilated left lung. This indicates that ischemia and nonventilation without inflation of the lung induces an increase in pulmonary compliance.

The injury process in the right lung started from 1 hour after warm ischemia-reperfusion of the left lung as soon as the process in the left lung started and continued for at least 3 hours after reperfusion. The pulmonary function did not recover, but gradually got worse during the experiment. No recovery process of either lung was observed in the acute phase of the experimental model. Therefore, it is necessary to elongate the observation period after reperfusion or shorten the warm ischemic period to observe the recovery process.

In this study, the levels of cellular adenine nucleotides and EC in the right lung showed no difference between the two groups, and the levels of ATP and EC in the left lung were significantly less in group I than in group II. These results indicate that ischemic injury is related to the metabolism of adenine nucleotides but reperfusion injury itself is not related to it during 3 hours after reperfusion.

There have been very few studies on the metabolism of adenine nucleotides in the lung. Date and associates [18] reported that in a canine left lung allotransplantation model, ATP levels were stable for up to 18 hours after cold (4° and 10°C) preservation, and a higher preservation temperature (22°C) caused a 28% decline in the ATP level after 18 hours of preservation. During the period of reperfusion, ATP levels decreased. Hall and colleagues [19] reported that cold (4°C) preservation of the lung for 120 minutes sustained a significantly higher energy state, and short periods of warm (36°C) ischemia for 30 minutes resulted in a significant depletion of the energy state, which may be a component of pulmonary injury during harvesting and preservation.

Protein concentration and phosphorus concentration of organized phospholipid in BALF were assessed. The protein concentration indicates the degree of the cellular membrane permeability of the pulmonary alveolar epithelium and endothelium, and the phosphorus concentration indirectly indicates the level of pulmonary surfactant. The protein concentration in BALF of the right lung at 210 minutes after the 90-minute period showed a greater level in group I than in group II. This indirectly indicates that reperfusion after 90 minutes of warm ischemia of the left lung causes an increase in membrane permeability of pulmonary alveolar epithelium and endothelium but no change in the surfactant system. The increase in membrane permeability results in pulmonary edema. Extravascular lung water of the right lung was greater in group I.

We make the following conclusions: (1) Ischemia-reperfusion of the left lung affects the nonischemic right lung. (2) The effects are the following: PaO₂ decreases, and P(A-a)O₂, pulmonary arterial resistance, and extravascular lung water increase. The degree of the effect is the same as that of the left ischemia-reperfusion lung at 3 hours after reperfusion. (3) The effect started as soon as the left lung injury started at 1 hour after reperfusion.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Address reprint requests to Dr Watanabe, Second Department of Surgery, Sapporo Medical University and Hospital, South 1, West 16, Chuo-ku, Sapporo 060, Japan.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 

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