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Meeting Abstracts  |   October 2006
Desflurane-induced Preconditioning against Myocardial Infarction Is Mediated by Nitric Oxide
Author Affiliations & Notes
  • Thorsten M. Smul, M.D.
    *
  • Markus Lange, M.D.
    *
  • Andreas Redel, M.D.
    *
  • Natalie Burkhard, B.S.
  • Norbert Roewer, M.D., Ph.D.
  • Franz Kehl, M.D., Ph.D., D.E.A.A.
    §
  • * Research Fellow, ‡ Professor of Anesthesiology and Chairman, § Professor of Anesthesiology and Vice-Chairman, Department of Anesthesiology, † Graduate Student, Department of Medicine.
Article Information
Meeting Abstracts   |   October 2006
Desflurane-induced Preconditioning against Myocardial Infarction Is Mediated by Nitric Oxide
Anesthesiology 10 2006, Vol.105, 719-725. doi:
Anesthesiology 10 2006, Vol.105, 719-725. doi:
VOLATILE anesthetics induce myocardial preconditioning through signal transduction pathways that are remarkably similar to those observed during ischemic preconditioning (IPC).1,2 Adenosine receptors,3–5 protein kinase C,4,6 inhibitory guanine nucleotide binding proteins,7 mitochondrial and sarcolemmal adenosine triphosphate–regulated potassium channels,3,7–9 reactive oxygen species (ROS),10–14 and reactive nitrogen species (RNS)13 have been implicated both in anesthetic-induced preconditioning (APC) and IPC. APC and IPC are characterized by an early (minutes)15–18 and a delayed (24 h)19,20 phase of preconditioning. The role of ROS and RNS as trigger or mediator of early and delayed IPC21–24 and APC,13,20 respectively, is under intense investigation. Novalija et al.  administered inhibitors of nitric oxide synthase (NOS) and ROS scavengers during ischemia/reperfusion injury in isolated hearts and demonstrated the essential role of ROS and RNS in early APC.1,13 For detailed review, see Yellon and Downey1 and Tanaka et al.  2 
Isoflurane11,12 and sevoflurane13 provide preconditioning against myocardial infarction by generation of ROS/RNS during early preconditioning. There is evidence that nitric oxide plays an important role in desflurane-induced preconditioning as well.25 However, it is unclear whether nitric oxide acts as a trigger or a mediator for desflurane-induced preconditioning in early preconditioning. Therefore, we tested whether nitric oxide is a trigger or a mediator of desflurane-induced preconditioning in vivo  by inhibiting nitric oxide formation by blockade of nitric oxide synthase either during (trigger phase) or after (mediator phase) administration of desflurane.
Materials and Methods
All experimental procedures and protocols used in this investigation were reviewed and approved by the regional governmental Animal Care and Use Committee government of Lower Franconia, Wuerzburg, Bavaria, Germany, and conformed with the regulations of the German Animal Protection Law.
General Preparation
Male New Zealand White rabbits (2.3–3.3 kg) were anesthetized with intravenous sodium pentobarbital (infusion of 30 mg/kg) via  the left auricular marginal vein after pretreatment with a eutectic mixture of lidocaine and prilocaine cream (EMLA; AstraZeneca, Wedel, Germany). Sufficient anesthesia was maintained with 20–30 mg/kg/h pentobarbital and was assured by recurrent testing of palpebral reflexes and hind-paw withdrawal throughout the experiment. After a ventral midline incision of the neck, a surgical tracheotomy was performed and the trachea was cannulated with a steel cannula. The animals were mechanically ventilated (Cicero; Draeger, Lübeck, Germany) using an air–oxygen mixture (70% air–30% oxygen). A 2.5-French microtipped catheter (Millar Instruments Inc., Houston, TX) was inserted via  the right femoral artery into the aorta for monitoring mean arterial pressure. Arterial blood gas tensions were regularly monitored via  a catheter in the auricular artery using an ABL 505 blood gas analyzer (Radiometer, Copenhagen, Denmark). Arterial blood gas tensions and acid–base status were maintained within a normal physiologic range by adjusting the respiratory rate and tidal volume, respectively. Rectal body temperature was maintained at 38.5°± 0.5°C26 by a servo-controlled heating pad (Föhr Medical Instruments, Seeheim, Germany).
Normal saline (0.9%) was administered as maintenance fluid throughout the experiment at a rate of 15 ml · kg−1· h−1. After a left fourth thoracotomy, a pericardiotomy was performed, and a 4-mm ultrasound probe (Transonic, Ithaca, NY) was placed around the pulmonary artery for measurement of cardiac output. Halfway between the base and the apex of the heart, a silk ligature (2-0) was placed around a prominent branch of the left anterior descending coronary artery to form a snare. By tightening the snare, a coronary artery occlusion (CAO) was produced, and reperfusion was instituted by loosening the snare.
Each rabbit received heparin (300 U/kg) 5 min before CAO for anticoagulation. CAO was verified by the presence of visual inspected epicardial cyanosis, regional dyskinesia in the ischemic zone, and electrocardiographic changes. Adequate reperfusion was confirmed by epicardial hyperemic response and reversion of electrocardiographic changes.
Hemodynamic parameters, body temperature, and the electrocardiogram were continuously recorded by an analog–digital acquisition board interface (Data Translation, Marlboro, MA) and a personal computer. The data were analyzed by commercially available hemodynamic data analysis software (Notocord-hem 3.5; Notocord Systems, Croissy-sur-Seine, France). Data were digitized at a sampling rate of 1,000 Hz.
Experimental Protocol
The experimental design of this study is illustrated in figure 1. Thirty minutes after instrumentation and calibration were completed, baseline systemic hemodynamics were recorded. Rabbits were randomly assigned to five groups. All rabbits were subjected to 30 min of CAO followed by 3 h of reperfusion. The animals received either 0.0 or 1.0 minimum alveolar concentration (MAC) desflurane (Baxter Deutschland GmbH, Heidelberg, Germany) for 30 min, which was discontinued 30 min before CAO (memory period). In separate experimental groups, the nonselective NOS inhibitor N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1for 10 min) was given intravenously either 20 min before or 10 min after desflurane. l-NA was dissolved in 10 ml normal saline (0.9%) as vehicle.
Fig. 1. Experimental protocol. All animals were subjected to 30 min of coronary artery occlusion (CAO) followed by 3 h of reperfusion. Desflurane was administered for 30 min and discontinued 30 min before CAO at 0.0 (control) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). l-NA was dissolved in 10 ml normal saline (0.9%) as vehicle. All animals received either l-NA or vehicle (VEH) intravenously. 
Fig. 1. Experimental protocol. All animals were subjected to 30 min of coronary artery occlusion (CAO) followed by 3 h of reperfusion. Desflurane was administered for 30 min and discontinued 30 min before CAO at 0.0 (control) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). l-NA was dissolved in 10 ml normal saline (0.9%) as vehicle. All animals received either l-NA or vehicle (VEH) intravenously. 
Fig. 1. Experimental protocol. All animals were subjected to 30 min of coronary artery occlusion (CAO) followed by 3 h of reperfusion. Desflurane was administered for 30 min and discontinued 30 min before CAO at 0.0 (control) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). l-NA was dissolved in 10 ml normal saline (0.9%) as vehicle. All animals received either l-NA or vehicle (VEH) intravenously. 
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In a separate group, l-NA was given alone 80 min before CAO, thus corresponding to the time point 20 min before desflurane. All animals received either l-NA or vehicle intravenously 80 min before CAO. The infusion rate of 1.3 mg · kg−1· min−1l-NA was chosen according to the results of a study by Bolli et al.  27 showing that a total dose of 13 mg/kg l-NA inhibited stimulated release of nitric oxide but did not alter basal vascular tone by a decrease of basal endothelial nitric oxide release and did not affect hemodynamics in conscious rabbits, although it is sufficient to block induced endothelial NOS activity. The MAC value used in the present investigation was 8.9 vol%.28 End-tidal concentrations of desflurane were measured continuously with a calibrated infrared anesthetic analyzer (Draeger, Lübeck, Germany).
At the end of the experimental protocol, the area at risk for infarction (AAR) was determined by infusion of 2 ml patent blue (0.1 g/ml; Sigma-Aldrich, Taufkirchen, Germany), after reocclusion of the coronary artery. The rabbits were killed with a lethal dose of pentobarbital, and the heart was rapidly excised. The left ventricle was separated from both atria and right ventricle. The left ventricle was cut into five slices from apex to base. The ischemic zone, i.e.  , the nonstained red myocardium (AAR), was separated from the normal nonischemic zone of blue-stained left ventricular myocardium. For determination of myocardial infarct size, the myocardial samples were incubated at 37°C for 20 min in 1% 2,3,5-triphenyltetrazolium chloride in 0.1 m phosphate buffer adjusted to pH 7.4. The tetrazolium salt is reduced by mitochondrial enzymes to a brick red, lipid-soluble formazan. Viable tissue therefore stains brick red, whereas the infarct appears pale white.29 After overnight storage in 10% formaldehyde, normal zone, ischemic zone, and infarct size were gravimetrically determined by a blinded investigator. Infarct size was expressed as percentage of the ischemic zone. Because the relation between infarct size and AAR has a nonzero intercept, an AAR of at least 15% was required to minimize the likelihood that risk zone size alone could influence the results.30 Therefore, rabbits with an AAR less than 15% of left ventricular mass and those that developed intractable ventricular fibrillation were excluded from subsequent analysis.
Measurement of NOS Activity
Nitric oxide synthase activity was measured by the rate of conversion of [3H]l-arginine to [3H]l-citrulline (NOSdetect Assay Kit; Stratagene, La Jolla, CA). Fifteen animals were randomly assigned to additional three experimental groups (n = 5). The animals were anesthetized as described before and killed 30 min and 5 min after 1.0 MAC desflurane administration for 30 min, respectively. Left ventricular tissue samples were obtained, snap frozen with liquid nitrogen, and stored at −80°C. Tissue samples (150 mg) were homogenized in 10 volumes of 1× homogenization buffer (pH 7.4, 250 mm Tris-HCl, 10 mm EDTA, 10 mm EGTA) on ice. After centrifugation at 15,000 rpm for 5 min at 4°C, 10 μl supernatant for each sample was incubated during 30 min at 37°C in 40 μl reaction mixture (25 μl of 2× reaction buffer; 5 μl reduced nicotinamide adenine dinucleotide phosphate, 10 mm; 1 μl [3H]l-arginine, 1 μCi/μl; 5 μl CaCl2, 6 mm; 4 μl dH2O). The reaction was terminated by adding 400 μl stop buffer (50 mm HEPES, 5 mm EDTA) at room temperature. Equilibrated resin was added to the solution to bind [3H]l-arginine. [3H]l-citrulline was separated by centrifugation through spin cups. Radioactivity of [3H]l-arginine and [3H]l-citrulline was quantified by liquid scintillation spectroscopy in counts per minute. NOS activity is expressed as ratio of radiolabeled l-citrulline to l-arginine.
Statistical Analysis
Statistical analysis of data within and between groups was performed with analysis of variance for repeated measures followed by the Duncan test (StatMost 3.6 for Windows; Dataxiom Software Inc., Los Angeles, CA). Changes within and between groups were considered statistically significant when the P  value was less than 0.05. All data are expressed as mean ± SEM.
Results
Forty-two rabbits were instrumented to obtain 39 successful experiments. Three rabbits were excluded because of intractable ventricular fibrillation (1 l-NA, 1 DES + l-NA, 1 l-NA + DES). For determination of NOS activity, 15 additional rabbits were anesthetized to obtain 14 successful experiments. A single rabbit was excluded because of intractable ventricular arrhythmia during desflurane administration.
Systemic Hemodynamics
There were no differences in baseline hemodynamics between groups (table 1). The heart rate was stable throughout the experiments. Desflurane significantly decreased mean arterial pressure, which returned to baseline values within 5 min after discontinuation of desflurane. There were no significant differences in heart rate or mean arterial pressure among groups during and after coronary artery occlusion. Administration of l-NA either before or after desflurane had no significant effect on hemodynamic parameters.
Table 1. Systemic Hemodynamics 
Image not available
Table 1. Systemic Hemodynamics 
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Myocardial Infarct Size
Body weight, left ventricular mass, AAR, and the ratio of AAR to left ventricular mass were similar between groups (table 2). Desflurane significantly reduced myocardial infarct size from 56 ± 8% (n = 8) in control experiments to 35 ± 4% (n = 8) as illustrated in figure 2. Desflurane-induced preconditioning was totally blocked by l-NA given either during or after desflurane inhalation, and infarct sizes were 58 ± 4% (n = 8) and 59 ± 9% (n = 7), respectively. l-NA alone had no effect on infarct size (56 ± 7%; n = 8).
Table 2. Area at Risk 
Image not available
Table 2. Area at Risk 
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Fig. 2. Myocardial infarct size (IS) expressed as a percentage of the left ventricular area at risk (AAR). Desflurane was administered for 30 min and discontinued 30 min prior to coronary artery occlusion at 0.0 (CON) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. n = 8 in all groups except for DES + l-NA (n = 7). 
Fig. 2. Myocardial infarct size (IS) expressed as a percentage of the left ventricular area at risk (AAR). Desflurane was administered for 30 min and discontinued 30 min prior to coronary artery occlusion at 0.0 (CON) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. n = 8 in all groups except for DES + l-NA (n = 7). 
Fig. 2. Myocardial infarct size (IS) expressed as a percentage of the left ventricular area at risk (AAR). Desflurane was administered for 30 min and discontinued 30 min prior to coronary artery occlusion at 0.0 (CON) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. n = 8 in all groups except for DES + l-NA (n = 7). 
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Measurement of NOS Activity
The NOS activity assay detected a significant increase of NOS activity in the left ventricular tissue 5 min after desflurane administration (n = 5; 1.93 ± 0.2) compared with control animals (n = 5; 1.39 ± 0.11) as illustrated in figure 3. The NOS activity assay revealed no significant difference between control animals and the preconditioned animals 30 min after desflurane administration (n = 4; 1.03 ± 0.18).
Fig. 3. Nitric oxide synthase (NOS) activity assay. NOS activity expressed as ratio of [3H]l-citrulline/[3H]l-arginine in left ventricular myocardial tissue 5 min (DES 5′; n = 5) and 30 min (DES 30′; n = 4) after 30-min application of 1.0 minimum alveolar concentration desflurane, respectively. Control animals (CON; n = 5) received no desflurane. All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. 
Fig. 3. Nitric oxide synthase (NOS) activity assay. NOS activity expressed as ratio of [3H]l-citrulline/[3H]l-arginine in left ventricular myocardial tissue 5 min (DES 5′; n = 5) and 30 min (DES 30′; n = 4) after 30-min application of 1.0 minimum alveolar concentration desflurane, respectively. Control animals (CON; n = 5) received no desflurane. All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. 
Fig. 3. Nitric oxide synthase (NOS) activity assay. NOS activity expressed as ratio of [3H]l-citrulline/[3H]l-arginine in left ventricular myocardial tissue 5 min (DES 5′; n = 5) and 30 min (DES 30′; n = 4) after 30-min application of 1.0 minimum alveolar concentration desflurane, respectively. Control animals (CON; n = 5) received no desflurane. All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. 
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Discussion
Nitric oxide–dependent signaling plays a pivotal role in IPC.27 Several studies demonstrate that exogenous nitric oxide prolongs myocardial ischemic tolerance22,25,27,31 and reduces arrhythmias during ischemia and reperfusion.32 Administration of N  -nitro-l-arginine methyl ester (l-NAME) during the ischemic stimulus of delayed IPC blocks its beneficial effects, indicating the role of endogenous nitric oxide as a trigger of delayed ischemic-induced preconditioning.33 The role of nitric oxide is well studied regarding IPC.24 
In the current study, desflurane inhalation at 1.0 MAC for 30 min followed by a 30-min memory period produced a marked protection against myocardial infarction in rabbits. Blockade of NOS by l-NA given during either the trigger or the mediator phase of early preconditioning completely abolished its cardioprotective effect. Therefore, the current data indicate that in early desflurane-induced preconditioning formation of nitric oxide by NOS plays an important role.
All three isoforms of NOS are inhibited by l-NA: neuronal NOS, inducible NOS, and endothelial NOS (eNOS).34,35 Neuronal NOS is detectable at only low levels in both ventricles.34,36 Although inducible NOS is absent in cardiomyocytes under physiologic conditions, it is expressed in these cells after stimulation, e.g.  , by pacing-induced heart failure in rabbits,37 or in delayed IPC.38 In the normal myocardium, eNOS is clearly the predominant isoform of NOS and is expressed in coronary microvessels36 and cardiomyocytes.39 Cardiomyocytes express eNOS starting from early embryonic stages and generate nitric oxide under normal physiologic conditions.40–42 Therefore, the most likely source for nitric oxide in normal hearts is formation by eNOS. Chiari et al.  20 detected increased levels of eNOS messenger RNA in rabbits 2 and 24 h after isoflurane administration. Higher expression of eNOS was demonstrated in immunohistochemistry studies in myocardial microvessels. Inducible NOS expression was not affected by isoflurane in rabbits in the early APC time frame.20 The fact that inhibition of NOS abrogated early desflurane-induced preconditioning implies that volatile anesthetics have stimulating effects on nitric oxide generation. In a study by Baumane et al.  43 using electron paramagnetic resonance spectroscopy, it was demonstrated that l-NA abolished the increase of nitric oxide content in the rat brain induced by volatile anesthetics.
In the current study, NOS activity as measured by l-arginine/l-citrulline conversion assay was significantly increased immediately after desflurane application and declined to normal level 30 min after desflurane administration. According to this finding, it is less likely that a de novo  synthesis of NOS causes early APC, because upraising NOS expression would increase nitric oxide formation for more than 5 min as shown for eNOS in delayed APC.20 In conclusion, the observed desflurane-induced increase of eNOS activity is the most likely source of nitric oxide for triggering signaling cascades of desflurane-induced preconditioning.
In isolated guinea pig hearts, volatile APC has been shown to be mediated by formation of nitric oxide–dependent reactive intermediate peroxynitrite.13 At which targets nitric oxide or its reactive intermediates finally act is unknown. Nitric oxide is known to influence various intracellular mechanisms partially independently, e.g.  , adenosine triphosphate–regulated potassium channels44 or cyclooxygenase 2.38 In addition, nitric oxide has the ability to attenuate deleterious free radical actions itself.45 Therefore, it remains unclear where exactly nitric oxide interacts in the signaling cascade of APC.
One aim of our study was to investigate whether nitric oxide acts as a trigger or a mediator within the signaling cascade of early volatile APC. A trigger in the preconditioning paradigm is characterized as an event that initiates the transformation of myocardial tissue into the preconditioned state. Once triggered, this state is maintained, although the trigger is removed. Therefore, application of an inhibitor during ischemia or reperfusion is not able to block preconditioning, because downstream mediators and effectors are already activated. The current results, where blockade of NOS during the mediator phase abrogated desflurane-induced preconditioning, support the conclusion that nitric oxide serves the role as a mediator of desflurane-induced preconditioning.
The current results must be interpreted within the constraints of several potential limitations. l-NA is a competitive substrate analogon of l-arginine and specifically inhibits NOS isoenzymes.46 However, the possibility that l-NA may have affected other signaling cascades or proteins involved in myocardial protection cannot be completely excluded from the analysis.
Our results further demonstrate that l-NA given during the trigger phase also abrogated preconditioning. After l-NA administration, the inhibition of NOS is effective during the mediator phase, because NOS blockade lasts up to 6 h.47 Unfortunately, there are no short-acting NOS inhibitors available. Therefore, it cannot be excluded that nitric oxide might in addition serve as a trigger. On the other hand, it can be excluded that nitric oxide acts solely as a trigger in desflurane-induced preconditioning, because then l-NA given during the mediator phase (i.e.  , after desflurane) would not have affected desflurane-induced preconditioning. Thereby it can be concluded that nitric oxide acts as a mediator of desflurane induced early preconditioning, but an additional role as a trigger cannot be excluded on the basis of the current data in vivo  .
Myocardial infarct size is determined primarily by the size of the AAR and extent of coronary collateral perfusion. However, in the current investigation, AAR expressed as a percentage of total left ventricular mass was similar between groups. Rabbits have been shown to possess little if any coronary collateral blood flow.48 Therefore, it seems unlikely that differences in collateral perfusion between groups account for the observed results. The rate–pressure product was stable throughout the experimental time course in all groups. Therefore, the reduction in myocardial infarct size produced by desflurane-induced preconditioning occurred independent of changes of major determinants of myocardial oxygen consumption. However, coronary venous oxygen consumption was not calculated in the current study. Therefore, it cannot be ruled out that hemodynamic differences could influence infarct size.
In summary, the current results confirm that early preconditioning with the volatile anesthetic desflurane produces a marked reduction in infarct size in an in vivo  myocardial infarction rabbit model even with a memory period of 30 min. Inhibition of desflurane-induced NOS activation by l-NA eliminated early preconditioning by this volatile agent, indicating that nitric oxide acts as a mediator of early APC.
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Fig. 1. Experimental protocol. All animals were subjected to 30 min of coronary artery occlusion (CAO) followed by 3 h of reperfusion. Desflurane was administered for 30 min and discontinued 30 min before CAO at 0.0 (control) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). l-NA was dissolved in 10 ml normal saline (0.9%) as vehicle. All animals received either l-NA or vehicle (VEH) intravenously. 
Fig. 1. Experimental protocol. All animals were subjected to 30 min of coronary artery occlusion (CAO) followed by 3 h of reperfusion. Desflurane was administered for 30 min and discontinued 30 min before CAO at 0.0 (control) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). l-NA was dissolved in 10 ml normal saline (0.9%) as vehicle. All animals received either l-NA or vehicle (VEH) intravenously. 
Fig. 1. Experimental protocol. All animals were subjected to 30 min of coronary artery occlusion (CAO) followed by 3 h of reperfusion. Desflurane was administered for 30 min and discontinued 30 min before CAO at 0.0 (control) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). l-NA was dissolved in 10 ml normal saline (0.9%) as vehicle. All animals received either l-NA or vehicle (VEH) intravenously. 
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Fig. 2. Myocardial infarct size (IS) expressed as a percentage of the left ventricular area at risk (AAR). Desflurane was administered for 30 min and discontinued 30 min prior to coronary artery occlusion at 0.0 (CON) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. n = 8 in all groups except for DES + l-NA (n = 7). 
Fig. 2. Myocardial infarct size (IS) expressed as a percentage of the left ventricular area at risk (AAR). Desflurane was administered for 30 min and discontinued 30 min prior to coronary artery occlusion at 0.0 (CON) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. n = 8 in all groups except for DES + l-NA (n = 7). 
Fig. 2. Myocardial infarct size (IS) expressed as a percentage of the left ventricular area at risk (AAR). Desflurane was administered for 30 min and discontinued 30 min prior to coronary artery occlusion at 0.0 (CON) or 1.0 minimum alveolar concentration desflurane (DES). The nonselective nitric oxide synthase inhibitor  N  ω-nitro-l-arginine (l-NA; infusion rate 1.3 mg · kg−1· min−1) was given as a 10-min intravenous infusion before (l-NA + DES, trigger phase) or after desflurane (DES + l-NA, mediator phase) or alone (l-NA). All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. n = 8 in all groups except for DES + l-NA (n = 7). 
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Fig. 3. Nitric oxide synthase (NOS) activity assay. NOS activity expressed as ratio of [3H]l-citrulline/[3H]l-arginine in left ventricular myocardial tissue 5 min (DES 5′; n = 5) and 30 min (DES 30′; n = 4) after 30-min application of 1.0 minimum alveolar concentration desflurane, respectively. Control animals (CON; n = 5) received no desflurane. All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. 
Fig. 3. Nitric oxide synthase (NOS) activity assay. NOS activity expressed as ratio of [3H]l-citrulline/[3H]l-arginine in left ventricular myocardial tissue 5 min (DES 5′; n = 5) and 30 min (DES 30′; n = 4) after 30-min application of 1.0 minimum alveolar concentration desflurane, respectively. Control animals (CON; n = 5) received no desflurane. All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. 
Fig. 3. Nitric oxide synthase (NOS) activity assay. NOS activity expressed as ratio of [3H]l-citrulline/[3H]l-arginine in left ventricular myocardial tissue 5 min (DES 5′; n = 5) and 30 min (DES 30′; n = 4) after 30-min application of 1.0 minimum alveolar concentration desflurane, respectively. Control animals (CON; n = 5) received no desflurane. All data are mean ± SEM. * Significantly (  P  < 0.05) different from control animals. 
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Table 1. Systemic Hemodynamics 
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Table 1. Systemic Hemodynamics 
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Table 2. Area at Risk 
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Table 2. Area at Risk 
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