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Ann Thorac Surg 2000;70:1684-1689
© 2000 The Society of Thoracic Surgeons

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Original articles: general thoracic

Ascorbic acid for amelioration of reperfusion injury in a lung autotransplantation model in sheep

^a Department of Thoracic and Cardiovascular Surgery, University Hospitals, Homburg/Saar, Germany

Address reprint requests to Dr Demertzis, Cardiocentro Ticino, Via Tesserete 48, CH-6900 Lugano, Switzerland
e-mail: stef.dem{at}mailcity.com

	Abstract

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Background. Reperfusion injury is the leading cause of early graft dysfunction after lung transplantation. Activation of neutrophilic granulocytes with generation of free oxygen radicals appears to play a key role in this process. The efficacy of ascorbic acid as an antioxidant in the amelioration of reperfusion injury after lung transplantation has not been studied yet.

Methods. An in situ autotransplantation model in sheep is presented. The left lung was flushed (Euro-Collins solution) and reperfused; after 2 hours of cold storage, the right hilus was then clamped (group R [reference], n = 6). Group AA animals (n = 6) were treated with 1 g/kg ascorbic acid before reperfusion. Controls (group C, n = 6) underwent hilar preparation and instrumentation only.

Results. In group R, arterio-alveolar oxygen difference (AaDO₂) and pulmonary vascular resistance (PVR) were significantly elevated after reperfusion. Five of 6 animals developed frank alveolar edema. All biochemical parameters showed significant PMN activation. In group AA, AaDO₂, PVR, work of breathing, and the level of PMN activation were significantly lower.

Conclusions. The experimental model reproduces all aspects of lung reperfusion injury reliably. Ascorbic acid was able to weaken reperfusion injury in this experimental setup.

	Introduction

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Lung transplantation has evolved into a clinically accepted treatment option for patients with end-stage pulmonary disease. One of the leading causes of early morbidity and mortality, however, is early graft dysfunction. The factors leading to this condition include unrecognized donor pathology, inadequate preservation, infection, and pathologic changes occurring during reperfusion, generally termed reperfusion injury.

The clinical picture of reperfusion injury includes increased pulmonary vascular resistance, interstitial and alveolar pulmonary edema, and impaired gas exchange. In addition, we have observed systemic vasodilatation and intravascular hypovolemia in the face of "third space loss", indicating a systemic inflammatory component of this process. The exact mechanism of reperfusion injury is not completely understood, but it appears that generation of free oxygen radicals and activation of polymorphonuclear neutrophilic granulocytes (PMN) play an important role in its development.

Experimental transplantation after cold storage has been so far unable to duplicate the complete clinical picture of reperfusion injury, ie, not only hypoxia but also severe impairment of endothelial permeability including the development of frank alveolar edema. We have developed an experimental model of unilateral autotransplantation of the lung in sheep. Using flush perfusion with modified Euro-Collins solution and cool storage, we have been able to reproduce not only impaired gas exchange and elevated pulmonary vascular resistance, but also local and systemic aspects of the reperfusion syndrome, ie, interstitial and alveolar pulmonary edema, and the syndrome of generalized capillary leak. In order to elucidate the role of free oxygen radicals in this setting, we studied the effect of ascorbic acid—a potent antioxidant—in ameliorating lung reperfusion injury in this model of lung autotransplantation in sheep.

	Material and methods

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A model of left lung in situ autotransplantation combined with hilar stripping was designed for these experiments.

Female Merino sheep with a body weight between 25 and 35 kg were used for the experiments. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society of 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 (National Institutes of Health Publication No 80-23, revised 1978). The experimental protocol was approved by the local regulatory authorities.

Anesthesia
The animals were premedicated with azaperon (200 mg, Stresnil; Janssen, Neuss, Germany) and atropine sulfate (0.5 mg; B. Braun, Melsungen, Germany) both given by intramuscular (IM) injection. A venous line was established by puncturing an auricular vein. Induction of anesthesia was performed with sodium thiopental (250 mg intravenously [IV], Trapanal; Byk Gulden, Konstanz, Germany). The animals were intubated with an orotracheal tube (8.5 mm internal diameter; Rüsch, Germany). Volume-controlled mechanical ventilation was instituted (Siemens Servo 900 C respirator; Siemens, Erlangen, Germany). Initially tidal volume was set to 10 mL/kg body weight, respiratory rate to 14 breaths per minute with an FiO₂ of 0.5. The respirator settings were subsequently adjusted to achieve a PCO₂ of 40 to 45 mm Hg and arterial oxygen saturation of more than 90%.

Anesthesia was maintained with sodium thiopental and fentanyl (300 mg, Trapanal; Byk Gulden, Konstanz, Germany; and 0.2 mg, Fentanyl; Janssen, Neuss, Germany) both given as IV bolus injections every 15 to 30 minutes. For muscular relaxation pancouronium bromide (4 mg IV, Pancuroniumbromid; Organon Teknika, Eppelheim, Germany) was added as appropriate. The experiment was terminated by IV injection of T61 (10 mL; Hoechst, Frankfurt/Main, Germany).

Surgical technique
The animal was placed in the right lateral position. A central venous catheter was inserted in the left external jugular vein percutaneously using the Seldinger technique. An additional central venous port for subsequent placement of a Swan-Ganz catheter was introduced in the same fashion. An arterial catheter for invasive blood pressure monitoring was placed in the left carotid artery in the same fashion.

A lateral thoracotomy was performed in the left fourth intercostal space. The left main pulmonary artery and bronchus were isolated in the pulmonary hilum. The pericardium was opened and the origin of the right pulmonary artery dissected after preparing the pulmonary trunk bifurcation. Dissecting the space between the right pulmonary artery and tracheal bifurcation, a tape was passed around the right main bronchus (Fig 1). The pulmonary veins were dissected at their entrance into the left atrium. During these steps of the surgical procedure particular care was taken to minimize manipulation of the ventilated left lung.

View larger version (120K): [in this window] [in a new window]   Fig 1. Surgical field after complete preparation. Left main bronchus and right main bronchus, as well as left and right pulmonary arteries (LPA, RPA) are isolated. (AO = aorta; LB = left bronchus; RB = right bronchus.) *Tape around BR.

Heparin was given intravenously (300 IU/kg). A cannula was placed in the pulmonary artery and the artery was cross-clamped proximally. A side-biting clamp was placed on the left atrium central to the left pulmonary veins and an incision was made for fluid drainage. The left lung was then flushed with cold modified Euro-Collins solution (60 mL/kg). Pulmonary pressure was monitored during flushing and was kept between 12 and 15 mm Hg. Ventilation was continued during flush perfusion. After completion of perfusion, the main left bronchus was transsected between two vascular clamps with the lung kept semi-inflated. The lung was left in situ and covered with cold towels. The temperature was measured in the left interlobar space. When the temperature exceeded 15°C, additional cold saline was applied to the towels.

Total ischemic time of the left lung was set at 2 hours. The transsected left bronchus was reconstructed at the end of the ischemic period with a continuous 4-0 Prolene suture (Ethicon, Hamburg, Germany) with the left lung deflated. The incision of the left atrium was closed and reperfusion begun with removing the clamp from the pulmonary artery. Hemodynamics were allowed to achieve a steady state during the initial 15 minutes of reperfusion. Right pulmonary artery and right main bronchus were then occluded by vascular clamps, thus making the animal dependent on the left lung only.

Experimental protocol
Cardiopulmonary assessment
Assessment of cardiopulmonary function consisted of the following: measurement of cardiac output by thermodilution (Cardiac Output Computer; Hoyer, Bremen, Germany); registration of pulmonary artery, left atrial, central venous, and arterial pressures; measurement of arterial blood gases (Blood Gas Analyzer Model 178; CIBA Corning, Oberlin, OH); and measurement of work of breathing ([WOB] Bicore CP 100 Pulmonary Monitor Computer; Bicore, Irvine, CA).

Subsequently the arterio-alveolar oxygen difference (AaDO₂) and the pulmonary vascular resistance (PVR) were calculated according to the following formulas:

where P_bar is barometric pressure, P_H2O is partial pressure of water vapor, FIO₂ is inspiratory oxygen fraction, and PaO₂ and PaCO₂ are arterial partial pressures for O₂ and CO₂). where MPAP is pulmonary artery mean pressure, LAP is left atrial pressure, and CO is cardiac output.

The evaluation time points were at the start of the experiment, after completion of instrumentation and hilar preparation, and 60, 120, and 180 minutes after reperfusion.

After termination of the experiment the total volume of infused fluids was calculated, as documented in the anesthesia protocol.

Biochemical assessment of leukocyte activation
The zymosan-induced and luminol-enhanced chemiluminescence (ZE-CL) of isolated polymorphonuclear granulocytes (PMN) was determined in a six-channel Biolumat LB 9505 (Bethold, Wildbad, Germany) according to standard described methods [1]. Peak maximum values were measured for isolated granulocyte suspensions, corrected for the corresponding blanks by substraction, and normalized to 25,000 cells. The PMN granulocytes were isolated from citrate-blood.

The activity of lung tissue myeloperoxidase (MPO) was determined photometrically [2] with H₂O₂ and o-dianisidine dihydrochloride as substrates. One unit of myeloperoxidase was defined as the activity that catalyzed the reaction of 1 µmol of H₂O₂ per minute under the test conditions employed. MPO activity was measured in tissue specimens obtained from both right and left lungs before termination of the experiment. Tissue MPO activity was calculated after subtraction of the MPO activity corresponding to the blood content of the tissue specimen out of the total MPO activity of the specimen.

Experimental groups
Three experimental groups were studied. In group C (control) animals underwent thoracotomy, preparation of both hili and instrumentation as described above without ischemia and subsequent manipulations. In group R (reference) the complete in situ left lung autotransplantation was performed including flush perfusion and cold storage. In group AA (ascorbic acid) a high dose of vitamin C was administred just before and during the first 10 minutes of reperfusion (1 g/kg body weight). Each group consisted of 6 successful experiments.

Statistical analysis
Values are presented as means ± standard error of the mean (SEM). Comparisons of mean values among the 3 groups were performed by analysis of variance (ANOVA) for repeated measurements and subsequent post hoc tests (Bonferroni/Dunn). The p values were corrected for the 5% level of statistical significance. Nonparametric values were compared either with the Wilcoxon or the ² test. Analyses were performed with the SPSS/Mac statistical software (version 6.1; SPSS Inc, Chicago, IL).

	Results

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Cardiopulmonary assessment
Alveolar pulmonary edema
No animal developed alveolar pulmonary edema in group C. In group R, 5 out of 6 animals, and in group AA, 4 out of 6 animals developed frank pulmonary edema. The frequency of pulmonary edema in group C was significantly lower than the frequency of pulmonary edema in groups R and AA (p = 0.009), whereas the differences between groups R and AA were statistically not significant (p = 0.501).

Blood-gas exchange
The AaDO₂ in group C remained stable throughout the experiment ranging from 215 ± 86 to 258 ± 122 mm Hg (Fig 2). The AaDO₂ values in group R became higher after reperfusion (range 209 ± 48 to 510 ± 148 mm Hg). A similar course could be observed for the AaDO₂ values in group AA (range 170 ± 68 to 360 ± 48 mm Hg). There were statistically significant differences between groups C and R (p = 0.0005), as well as between groups R and AA (p = 0.0011). The differences between groups C and AA were not statistically significant (p = 0.862).

View larger version (20K): [in this window] [in a new window]   Fig 2. Values for the alveolo-arterial oxygen difference over time. (*Statistically significant difference between group R and both groups AA and C.)

View larger version (21K): [in this window] [in a new window]   Fig 3. Values for pulmonary vascular resistance over time. (*Statistically significant differences between group C and both groups R and AA.)

Work of breathing
The WOB values remained stable in the control group throughout the experiment, ranging from 1.92 ± 0.24 J to 2.07 ± 0.3 J (Fig 4). In both other groups all values increased after reperfusion ranging from 2.25 ± 0.2 J to 4.01 ± 0.84 J in the reference group and from 1.74 ± 0.18 J to 2.96 ± 0.43 J in the ascorbic acid group. Values in group AA were significantly lower than in group R (p = 0.0204) but not significantly different from group C.

View larger version (46K): [in this window] [in a new window]   Fig 4. Values for work of breathing over time. (*Statistically significant differences between group R and both groups C and AA.)

View larger version (20K): [in this window] [in a new window]   Fig 5. Values for the chemiluminescence of isolated PMN leukocytes over time.

View larger version (30K): [in this window] [in a new window]   Fig 6. Tissue myeloperoxidase (MPO) in the right lung (R) and left lung (L). Statistically significant differences: (*between groups R and AA; +between groups AA and C.)

	Comment

Top Abstract Introduction Material and methods Results Comment References

After a period of ischemia, restoration of blood flow is accompanied by local capillary leakage due to endothelial damage. Endothelial damage results in part from oxygen radicals, which are generated when tissue is reperfused. In the lung, oxygen radicals are generated not only by phagocytic cells (PMN, macrophages), but also by lung parenchymal cells such as endothelial cells [5]. One intracellular source of oxygen radicals is the enzyme xanthine oxidase. Superoxide produced by activated neutophils and macrophages initiates the alteration of xanthine dehydrogenase to an oxidase that produces hydrogen peroxide (H₂O₂). The alteration of xanthin dehydrogenase to oxidase can be also caused by activated complement components and cytokines [6]. Interestingly impairment of pulmonary function after ischemia of the lung has been shown not only in species with relatively high levels of xanthine oxidase, such as rats, but also in rabbit and sheep lungs, which are almost devoid of any significant xanthine oxidase activity. This can be explained by the alternative pathway of hydroxyl radicals formation. This is initiated by superoxide (produced by activated phagocytic cells), which reacts with nitric oxide (NO^.) to form peroxinitrous acid (ONOOH). This mechanism exists in cells with relatively high levels of NO^.-synthetase activity such as endothelial cells and macrophages [7].

The clinical and hemodynamic presentation of reperfusion syndrome in our experimental model was reliably reproducible. During reperfusion the function of the reperfused left lung declined continuously; AaDO₂ and PVR increased steadily. These values remained stable in the control group. The vast majority (5 out of 6) of animals undergoing the reference developed obvious alveolar edema; none of the lungs in the control group exhibited signs of alveolar edema. The model reproduced not only the organ specific manifestations of lung reperfusion injury but also the involvement of the whole organism. During reperfusion all animals required significantly more intravenous fluids than the controls to maintain hemodynamic stability. This is comparable with the clinical presentation of reperfusion injury and is in our opinion due to the generalized inflammatory reaction initiated by the reperfusion of the lung.

During respiratory burst highly activated PMN release their phagocytic granula after contact to activated or damaged cellular surface ("frustrane phagocytosis" [2]). The chemiluminescence value reflects the amount of intracellular phagocytic capacity of stimulated PMN. High values indicate unreleased granula and a rather low PMN activation state, whereas low values indicate that highly activated polymorphonuclear PMN just went through frustrane phagocytosis. Measurement of chemiluminescence is based on the amount of emitted photons, which is propotional to the amount of reactive oxygen metabolites built by the PMN for phagocytosis [8]. Enhancement with zymosane, which consists of parts of the starch cell membrane and stimulates the PMN by activating the complement system through the "alternative pathway," induces complete in vitro PMN degranulation for more accurate results.

The time course of the chemiluminescence values in our experiments suggests a profound PMN activation with ongoing reperfusion. This finding is compatible with those of several experimental studies, which demonstrated significant PMN activation during postischemic reperfusion [1, 8]. The secondary decline of the chemiluminescence values is most likely a sign of progressive PMN degranulation due to maximal activation. These results are supported by the measurements of MPO activity in lung tissue. MPO is present in the primary granula of PMN, the same granula as the PMN—specifically, elastase—and has been demonstrated to participate in the oxygen-dependent microbicidal activity of these cells. At the end of the experiment MPO activity in left lungs was significantly higher than in right lungs, which means that significant PMN invasion occurred into the left lung’s parenchyma.

Ascorbic acid is a potent antioxidant and oxygen free radical scavenger. It can easily reach both the extracellular and intracellular space owing to its small molecular size and is considered the most effective antioxidant in plasma [9, 10]. High doses of ascorbic acid (1 g/kg body weight) were able to improve oxygenation and pulmonary vascular resistance in a sheep endotoxin-induced adult respiratory distress syndrome model [11]. In other experimental studies ascorbic acid was found to effectively protect against oxidative reperfusion injury [9, 12]. In our experimental model we adopted the high dose of ascorbic acid (1 g/kg body weight).

The beneficial effect of ascorbic acid was partially reproducible in our investigation. Blood gas exchange in the ascorbic acid group improved compared with the reference group, and the difference reached statistical difference. With respect to PVR, no significant differences could be detected between the ascorbic acid and reference groups. The biochemical assessement of PMN activation revealed a mixed picture with respect to the role of ascorbic acid. Tissue MPO activity was significantly lower in the treated than in the reference group, suggesting lower levels of leukocytic activation in the treated animals. On the other hand the analysis of chemiluminescence did not reveal statistically significant differences between treated and reference animals.

This experimental model of left lung autotransplantation in sheep reproduces all aspects of the clinical picture of reperfusion injury. During reperfusion significant impairment of gas exchange and significant elevation of PVR could be documented. Obvious alveolar edema developed in the vast majority of animals undergoing the standard autotransplant procedure. In addition generalized inflammatory response became clinically obvious with the need of increased intravenous fluid requirements. Amelioration of pulmonary reperfusion injury by ascorbic acid has been feasible in this experimental setting; however, ascorbic acid does not seem to be the "golden shot" against reperfusion injury. Nevertheless, it adds to our armamentarium, since it is a natural, highly soluble, and nontoxic compound. An even higher dosage or a different application regime (eg, donor preischemic treatment) should be investigated in future studies.

	References

Top Abstract Introduction Material and methods Results Comment References

 

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