Interact CardioVasc Thorac Surg 2009;8:3-6. doi:10.1510/icvts.2008.176206
© 2009 European Association of Cardio-Thoracic Surgery
Work in progress report - Experimental |
Effect of systemically administered low potassium dextran solution on oxidative stress in a rat model of lung ischemia
Ronaldo Lopes Torresa,
Adriane Beló-Kleinb,
Cristiano Feijó Andradeb and
Paulo Francisco Guerreiro Cardosoc,*
a Department of Physiology, Federal University of Rio Grande do Sul-Porto Alegre, Rio Grande do Sul, Brazil
b Federal University of Rio Grande do Sul-Porto Alegre, Rio Grande do Sul, Brazil
c Department of Surgery, Division of Thoracic Surgery, Federal University of Health Sciences of Porto Alegre, Rio Grande do Sul, Brazil
Received 22 January 2008;
received in revised form 16 June 2008;
accepted 19 June 2008
*Corresponding author. Santa Casa de Porto Alegre-Pavilhao Pereira Filho Hospital, Rua Prof. Annes Dias 285 – 1andar, Porto Alegre, RS-90020-090, Brazil. Tel.: +55-51-32273909; fax +55-51-32282510.
E-mail address: cardosop{at}gmail.com (P.F.G. Cardoso).
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Abstract
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Systemic administration of the low-potassium dextran solution on the peripheral oxidative stress was evaluated in an animal model of lung ischemia-reperfusion in rats. In one experiment, male Wistar rats were divided into two groups (n=5): one received intravenous saline, whereas in the other the animals were given intravenous low potassium dextran solution. In another experiment, male Wistar rats were divided into four groups (n=5): control, ischemia, saline and low potassium dextran. Except for the control animals, all groups were submitted to left hilar clamping for 30 min, followed by reperfusion for 30 min. Saline or low potassium dextran was administered intravenously immediately before clamp removal. In the first experiment there were no significant differences in lipid peroxidation. Total radical trapping potential measurements showed a significant increase in animals receiving low potassium dextran; in the second experiment, there was an increase in lipid peroxidation in both saline and ischemia groups compared to controls, and low potassium dextran. Low potassium dextran group showed an increase in total radical trapping potential measurements compared to all other groups. Ischemia-reperfusion injury mediated by reactive oxygen species was attenuated by the systemic use of low potassium dextran in this animal model of ischemia-reperfusion of the lung.
Key Words: Ischemia-reperfusion; Free radicals; Low potassium dextran; Lung; Transplantation; Rats
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1. Introduction
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Pulmonary ischemia during donor organ retrieval and transplantation is associated with ischemia-reperfusion injury (I-R), resulting in endothelial cell damage and surfactant dysfunction [1]. Early lung allograft dysfunction which is closely related to I-R remains the most common cause of early mortality after lung transplantation, as well as a risk factor for bronchiolitis obliterans [2]. The I-R is characterized histopathologically by lung edema and neutrophil extravasation, and such changes in vascular permeability are mostly mediated by reactive oxygen species (ROS) acting during reperfusion. Studies on lung preservation focus on local effects following delivery of the preservation solution directly into the pulmonary circulation, using either antegrade or retrograde routes [3–5]. Little is known about the antioxidant properties of the lung preservation solutions and, to date, there have been no studies addressing the effects of the lung preservation solution administered systemically on oxidative stress. The objective of this study is to assess the potential effects of low potassium dextran (LPD) solution on oxidative stress when it is administered into the peripheral blood circulation. Such potential effects were then evaluated both in the presence and absence of lung ischemia.
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2. Materials and methods
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All animals received humane care in compliance with the ‘Guide for the Care and Use of Laboratory Animals’ (http://www.nap.edu/catalog/5140.html). This study was approved by the Ethics Committee of the University.
2.1. Experiment 1
Ten adult male Wistar rats (250–300 g) were anesthetized with intraperitoneal sodium penthotal (0.35 ml/100 g body weight) injection, followed by orotracheal intubation and mechanical ventilation (Harvard 683-Rodent Ventilator-Harvard Apparatus; FiO2=1.0, tidal volume=1 ml/100 g body weight, respiratory rate=65 bpm). Indwelling catheters were introduced into the femoral artery and vein. The animals were then randomly divided into two groups (n=5/group): intravenous (i.v.) injection of 0.5 ml of saline (SAL) and i.v. injection of LPD (12.5 ml/kg body weight). Peripheral blood samples were collected (0.5 ml/sample), via the femoral artery at the baseline (time 0), immediately (time 1), 15 min (time 2) and 30 min (time 3) after the administration of the solutions above. Oxidative stress was evaluated using the chemiluminescence (CL) technique, and the antioxidant defenses were determined by the total radical-trapping potential (TRAP) technique, which indicates the total antioxidant potential present in the plasma. For the CL technique, erythrocytes were utilized, whereas only plasma was utilized for TRAP.
2.2. Experiment 2
Twenty animals were anesthetized, intubated and canullated, using the same procedures as for experiment 1. Animals were randomly assigned into four groups (n=5/group): control (CON), ischemia (ISCH), saline (SAL), and LPD solution. Except for the animals in the CON group, all other groups were submitted to left hilar clamping for 30 min followed by reperfusion for 30 min. The arterial blood samples were obtained at time 0 (baseline=before thoracotomy), time 1 (immediately after clamp release), time 2 (15 min after reperfusion) and time 3 (30 min after reperfusion). In the CON group, samples were collected at the same time periods with neither thoracotomy nor clamping. Saline (0.5 ml) or LPD (12.5 ml/kg body weight) were administered intravenously at room temperature to animals in SAL and LPD groups, respectively, immediately before clamp removal. The lipid peroxidation was assessed through CL, and the antioxidant defenses through determination of the TRAP and the enzyme activities (catalase-CAT and superoxide dismutase-SOD). For the CAT and SOD activities and the CL, erythrocytes were utilized, whereas plasma was used for TRAP. Chemiluminescence was measured in a liquid scintillation counter (LKB Rack Beta Liquid Scintillation Spectrometer 1215, LKB – Produkter AB, Sweden) and data were expressed as counts/second/mg of hemoglobin (cps/mgHb). Plasma TRAP was measured by luminescence, using 2,2'-azo-bis(2-amidinopropane) (ABAP, a source of alkyl-peroxyl free radicals) and luminol and the results were expressed in µM Trolox. CAT activity was determined by following the decrease in hydrogen peroxide (H2O2) absorbance at 240 nm, and was expressed as pmol of H2O2 reduced/minute/mg of protein. SOD activity was expressed as units/mg of protein. Protein was measured using bovine serum albumin as standard. The conversion of hemoglobin to cyanomethaemoglobin by Drabkin reagent was measured against a standard curve. The values were expressed as mg/ml.
2.3. Statistical analysis
Results are expressed as mean±SEM. The differences among baseline groups were analyzed using Student's t-test or one-way ANOVA. The comparison of the effects along the different time periods was performed by repeated measures ANOVA, followed by Tukey's multiple comparisons test when needed. Results were analyzed as the percentage of the baseline for each animal (time 0=100%). Significance was set for P<0.05.
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3. Results
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3.1. Experiment 1
The comparison between the groups that received i.v. saline or preservation solution-LPD, showed neither significant difference in the CL measurement in baseline, nor there were there any differences over time or among the groups (Fig. 1). There were no significant differences in the TRAP measurements at baseline. When the effects were compared over time, a significant difference was found between the groups. Animals that received LPD, showed a significant increase in TRAP when compared with the CON animals group (27% increase; P=0.037). However, there were no differences over time, as well as no time–group interactions (Fig. 2).
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Fig. 1. Effect of systemic administration of saline or low potassium dextran (LPD) showing no significant differences between the groups and different time periods (P>0.05) on chemiluminescence (CL); CL values are expressed as mean±SEM of counts per second per milligram of hemoglobin (cps/mg Hb) (n=5/group) and represented as the percentage of baseline measurements.
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Fig. 2. Effect of systemic administration of saline or low potassium dextran (LPD) showing significant differences between the saline and LPD groups (*P=0.037) in total radical-trapping Potential (TRAP); CL values are expressed as mean±SEM of counts per second per milligram of hemoglobin (cps/mg Hb) (n=5/group); TRAP is expressed as mean±SEM of equivalents in µM trolox (n=5/group) and represented as the percentage of baseline measurements.
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3.2. Experiment 2
There were no significant differences among the groups for the CL, TRAP, CAT and SOD in baseline measurements. Comparing the measurements over time, there were significant differences among the four groups in CL (P=0.004). Although there were no significant differences between ISCH and SAL groups, both showed an increase in CL, when compared with the CON (17% increase; P=0.004) and the LPD animals (14% increase; P=0.018). No significant differences were found in animals in the CON and LPD groups (Fig. 3). Significant differences in TRAP were observed among the time periods (P=0.006), within the groups (P<0.0001), and with a significant time–group interaction (P<0.0001). The LPD group showed an increased TRAP in comparison with the other groups (20%, 27% and 11% increase compared to the control group, ischemia group and to the saline group, respectively) (Fig. 4). No significant differences were found in the activity of SOD and CAT enzymes.
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Fig. 3. Effects of the systemic administration of saline or LPD solutions upon CL and TRAP on the lung ischemia-reperfusion injury model: groups ischemia and saline showed significant differences when compared with the control (*P=0.004) and LPD animals (*P=0.018). No significant difference (#) was found in animals in the control group relative to the LPD group; CL values are expressed as mean±SEM of cps/mg Hb (n=5/group) and represented as the percentage of baseline measurements.
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Fig. 4. Effects of the systemic administration of saline or LPD solutions upon CL and TRAP on the lung ischemia-reperfusion injury model showing a significant increase in the LPD group in relation to the other groups (*P<0.0001); TRAP values are expressed as mean±SEM of equivalents µM trolox (n=5/group) and represented as the percentage of the baseline measurements.
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4. Discussion
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Reactive oxygen species (ROS) play an important role in primary graft failure. Oxidative stress is characterized by the formation of ROS, such as superoxide anion, hydrogen peroxide, and hydroxyl radical [6]. The generation of intra-cellular oxygen species has been found in the majority of lung cells, including the endothelial cells, type II alveolar epithelial cells, clara cells, ciliated airway epithelial cells and alveolar macrophages [7]. Despite abundant data on free radical scavengers given separately or incorporated into the solutions used for lung preservation, the antioxidant capacity of the preservation solutions themselves have not been addressed. The results of the present study suggest that the presence of LPD preservation solution in the systemic blood increases plasma's total antioxidant potential, both in the presence and absence of the lung ischemic event. It was also observed that there was a decrease in erythrocyte LPO in the presence of lung ischemia. In experiments 1 and 2, we found no differences between LPD and control groups in the lipid peroxidation, as evaluated by CL. Erythrocyte CL evaluated peripheral oxidative damage to membrane lipids, whereas TRAP showed whether the non-enzymatic antioxidants were altered by means of measuring the total antioxidant capacity of the plasma. In experiment 2 there was an increase in LPO in the animals submitted to lung ischemia, and it was not modified after the parenteral administration of saline. Conversely, it was modified by the administration of LPD solution, which caused a reduction of LPO to levels similar to those found in the control animals. The TRAP measurement in experiments 1 and 2 showed a significant increase in its values in the LPD group compared to the other groups. Such findings can be indicative of the intrinsic antioxidant properties of LPD, even when administered in the peripheral circulation at room temperature and at low doses that was approximately 25% of the dose administered into the pulmonary circulation during flushing. This dose was previously established by trial and error in the pilot study (unpublished data), in which higher doses were lethal to the animals. Conversely, the antioxidant enzymatic activities of CAT and SOD were similar when both groups and periods of time of measurement were compared. Since SOD and CAT represent the first step in the enzymatic detoxification of ROS, both are probably less sensitive to smaller changes in the production of ROS. This could explain the lack of differences between the groups in both enzyme concentrations.
Among five studies comparing LPD and Euro Collins solutions, in three the recipient oxygenation was improved and in four studies graft function was superior in the LPD group after lung tranplantation [8]. When LPD and Celsior soutions were compared, Celsior solution provided slightly superior endothelial preservation [1]. Yet, another study found no advantage of LPD as compared to modified Euro Collins solution in regards to early gas exchange, or impact on 1-year mortality [9]. Since LPD has been used in our clinical lung transplant program for a number of years, we embarked on studies to test newer ways of using the LPD solution. Although lower lipid peroxidation has been demonstrated in lungs preserved with LPD [10], the mechanism by which an extracellular low potassium solution reduces lung ischemia-reperfusion injury is not yet fully understood [11]. As opposed to the high potassium solutions, LPD solution does not cause membrane depolarization, allowing the cells to remain at near resting membrane potential. The LPD solution may, therefore, provide an appropriate ionic milieu that minimizes cell injury or activation, as indicated by the reduction of vasoconstriction or ROS production [11]. Although the pathway of ROS production is not yet completely understood, the use of a low potassium concentration in lung preservation solution seems to decrease the incidence of primary graft failure through the reduction of ROS production within the pulmonary vasculature [11]. The use of a free radical scavenger in addition to an extracellular solution has also proven successful. Sommer et al. [12] showed experimentally that myeloperoxidase activity was lowest when glutathione was added to LPD solution, yielding beneficial effects both on vascular function and surfactant composition in transplanted lungs. Glutathione has also been shown to be responsible for the efficacy of Celsior solution in lung preservation. Similarly, n-acetylcysteine, has been shown experimentally to protect the lungs from reperfusion injury after prolonged ischemia, possibly by attenuating post-transplant lung I-R [13].
Based on the findings of CL and TRAP measurements in the current experiments, we suggest that LPD itself may have an intrinsic antioxidant potential mediated either by one of its components or by the conjunction of the benefits of an extracellular solution altogether. On the other hand, LPD may be capable of inducing increased endogenous antioxidant potential by as yet unknown mechanisms. We acknowledge that the findings in our study must be taken with care, since the number of animals was small and this animal model is far fetched from the clinical setting. Nevertheless, the possible benefits of LPD given systemically at low doses, should be addressed in the future, in order to assess its potential application in lung transplantation. This can be particularly useful in an era in which, both the proliferation of transplant centers and organ shortage have pushed the transplant programs into marginal organ donation and post-mortem lung retrieval.
In conclusion, the present study suggests that ischemia-reperfusion injury mediated by ROS might be attenuated by the use of LPD itself given systemically. Such properties of LPD may play a role in its apparent ability in reducing graft failure. Since the potential benefits to the lung graft were not addressed in this study, future studies are required to assess the potential systemic antioxidant ability of LPD along with other lung preservation solutions in order to verify whether such features are specific for LPD or not.
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Acknowledgements
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The authors wish to thank: Iraci Torres, Maria B. Ferreira (Department of Physiology), Lucas Krieger Martins and Prof. S.A. Camey from the Institute of Mathematics, Department of Statistics of the Universidade Federal do Rio Grande do Sul; Vitrolife®-Sweden for providing the LPD (Perfadex®) used in the experiment.
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References
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Abstract
1. Introduction