1 Departments of Anesthesiology and Critical Care Medicine and 2 Cellular Biochemistry and Human Genetics, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
* Corresponding author: Department of Anesthesiology and CCM, Hadassah University Hospital, Jerusalem 91120, Israel. E-mail: gozaly{at}md.huji.ac.il
Accepted for publication May 15, 2005.
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Abstract |
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Methods. After i.v. administration of salicylate 100 mg kg1 and a 30 min stabilization period, New Zealand White rabbits were subjected to 40 min of regional myocardial ischaemia and 2 h of reperfusion. Ischaemic preconditioning was elicited by 5 min ischaemia followed by 10 min reperfusion (before the 40 min ischaemia). In another group, isoflurane (2.1%) was administered for 30 min, followed by 15 min washout, before the long ischaemia. Area at risk and infarct size were assessed by blue dye injection and tetrazolium chloride staining. We quantified the level of OH-mediated conversion of salicylate to its dihydrobenzoate derivatives (2,3- and 2,5-DHBAs). Normalized values of the DHBAs (ng DHBA per mg salicylate) were calculated.
Results. Mean (SE) infarct size was 57 (6)% of the risk area in the untreated controls. This was significantly smaller in the ischaemic preconditioning and isoflurane groups: 22 (5) and 23 (6)% respectively. At 10 min of reperfusion, ischaemic preconditioning limited the mean increase in 2,3-DHBA to 24% from baseline, compared with 81% in control and 74% in the isoflurane group. Normalized 2,5-DHBA was maximally increased by 75% in the untreated group, 4 min after reperfusion. Ischaemic preconditioning significantly inhibited this increase (24% increase from baseline, P<0.01). However, the increase observed in the isoflurane group was not different from control (71%).
Conclusions. As already known, ischaemic preconditioning and isoflurane markedly reduced infarct size. However, only ischaemic preconditioning decreased postischaemic production of hydroxyl radicals. These different effects suggest different protective mechanisms at the cellular level.
Keywords: anaesthetics volatile, isoflurane ; heart, ischaemia ; metabolism, free radicals ; model, rabbit
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Introduction |
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In contrast to their beneficial effect of starting the preconditioning cascade, a large number of studies have demonstrated that oxygen free radicals play a detrimental role in the pathogenesis of reperfusion injury, both directly by damaging membranes and enzymes and indirectly by initiating the inflammatory process.
We have reported that halothane administered before and during ischaemia in a canine model of ischaemia/reperfusion decreased hydroxyl radical (OH) formation significantly.7 When given during a sustained ischaemic period,8 or during 30 min followed by 15 min washout before prolonged ischaemia,9 halothane significantly decreased infarct size, thus inducing pharmacological preconditioning.
In the present study, performed in the in vivo rabbit model, we tested the hypothesis that ischaemic preconditioning and/or isoflurane would block postischaemic production of hydroxyl radicals in a similar manner to halothane. We used an experimental model in which hydroxyl radicals react with salicylate to generate 2,3- and 2,5-dihydroxybenzoic acids (DHBAs), which can be measured by a high-performance liquid chromatography with electrochemical detection. The formation of DHBAs after systemic administration of salicylate is used as an index of hydroxyl radical generation in the heart.
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Methods |
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General preparation
New Zealand White rabbits weighing between 2.5 and 3.5 kg were anaesthetized with i.v. pentobarbital 30 mg kg1 administered via a 20-G i.v. cannula in a marginal ear vein. Hetastarch 5 ml kg1 h1 was infused continuously via the i.v. cannula. Anaesthesia was maintained during the experiment by pentobarbital supplements as needed (according to pedal and palpebral reflexes). Isoflurane was used according to the study protocol. Neuromuscular blocking drugs were not administered in order to assess anaesthetic depth. The neck was opened with a ventral midline incision and a tracheostomy was performed. The rabbits' lungs were mechanically ventilated with positive pressure ventilation and an inspiratory fraction of oxygen of 1.0. The ventilation rate was 3035 b.p.m. and tidal volume was approximately 15 ml kg1. The respiratory rate was adjusted to keep the arterial pH in the physiological range of 7.357.45. End-expiratory carbon dioxide tension was monitored continuously. A 22-G catheter filled with heparinized saline was placed in a carotid artery for arterial blood pressure monitoring and blood sampling. Core body temperature was measured via a rectal temperature probe and maintained at 38.5 (0.3)°C (normothermic for rabbits) with radiant heat and a warming blanket. Needle electrodes were inserted subcutaneously in a lead II configuration to enable recording of an electrocardiogram (ECG) to determine heart rate and help confirm the occurrence of ischaemia (ST segment elevation) and reperfusion of the myocardium distal to the coronary occlusion. A left thoracotomy was performed in the fourth intercostal space and a 40 silk suture was passed around a prominent branch of the left coronary artery (approximately halfway between the apex and the base) and threaded through a small vinyl tube to form a snare. Coronary artery occlusion was achieved by tightening the snare around the coronary artery. Regional epicardial cyanosis and ST segment elevation in the ECG confirmed myocardial ischaemia. Reperfusion was achieved by releasing the snare and was confirmed by visual observation of reactive hyperaemia. Ventricular fibrillation, if it occurred, was reversed using direct mechanical stimulation: an index finger was flicked directly against the right ventricle side of the fibrillating heart one to three times to achieve defibrillation. Failure to convert to an organized rhythm after three attempts was defined as intractable fibrillation.
Experimental protocol
Before a 30-min stabilization period, all animals were given salicylate (100 mg kg1) i.v. Salicylate is a highly effective hydroxyl free radical scavenger, which, upon scavenging OH, forms 2,3- and 2,5-DHBA by hydroxylation. All animals underwent 40 min of regional ischaemia followed by 2 h of reperfusion. Preconditioning was elicited by 5 min of coronary occlusion followed by 10 min of reperfusion,10 beginning 15 min before the period of prolonged coronary occlusion. Rabbits were assigned randomly to the following groups according to a computer-generated random number schedule: control group (ischaemia and reperfusion without further intervention) (n=10); ischaemic preconditioning group (n=10) and isoflurane group (I, n=10). A 1.0 minimal alveolar concentration of isoflurane (2.1%)11 was started at the end of the stabilization period and administered for 30 min, followed by a 15 min washout period before coronary occlusion. End-tidal concentrations of isoflurane were measured continuously at the tip of the tracheostomy tube with a gas analyser (Drager Iris, Lübeck, Germany) that was calibrated with known standards before and during the experiments. Arterial pressure, heart rate and temperature were recorded continuously. Blood samples for hydroxyl free radical measurements were obtained as follows: at the beginning of the experiment (baseline), at 20 and 40 min of occlusion, every 2 min in the first 10 min of reperfusion, and thereafter at 20, 30, 60, 90 and 120 min of reperfusion.
We quantified the level of OH-mediated conversion of salicylate to its dihydroxybenzoate derivatives by high-performance liquid chromatography coupled with electrochemical detection. The results are expressed as the ratio of DHBA to salicylate concentration (ng DHBA per µg salicylate) to normalize DHBA levels to different concentrations of salicylate.7
Determination of infarct size and area at risk
At the end of the experimental protocol, hearts were excised, mounted on a Langendorff apparatus, and perfused with phosphate-buffered saline (PBS) at 100 cm H2O for 1 min to wash out intravascular blood. The coronary artery was re-occluded and methylene blue 0.2% was infused into the aortic root to label the normally perfused zone with deep blue colour, thereby delineating the risk zone as a non-stained area. The hearts were then removed from the Langendorff apparatus, trimmed of atria and great vessels, weighed and frozen (in a cold chamber at 18°C). Hearts were then cut into 2 mm transverse slices. The slices were incubated in 2,3,5-triphenyl tetrazolium chloride (TTC) 1% in pH 7.4 buffer for 20 min at 37°C. The slices were then placed in 10% neutral buffered formalin for 10 min to increase the contrast between stained and non-stained tissue. Since TTC stains viable tissue a deep red colour, non-stained tissue was presumed to be infarcted. Slices were then photographed, and risk and infarct areas in each slice were measured by computed planimetry. The mass-weighted average of the ratio of infarct area to the area at risk of the ventricle from each slice was determined (percentage infarction).
Quantification of hydroxyl radicals by interaction with salicylate
DHBA levels were identified and measured by high-performance liquid chromatography coupled with electrochemical detection using a Varian 5000 liquid chromatograph (Varian Medical Systems, Palo Alto, CA, USA), equipped with a Rheodyne 7125 sample injector (20 µl loop) (Rheodyne LLC, Rohnert Park, CA, USA). The column used for separation of salicylate and DHBA was a 25 cmx4 mm Li Chrospher 100 RP-18, 5 µm (E-Merck, Darmstadt, Germany). The mobile phase contained 0.03 M citric acid, 0.03 M acetic acid, sodium azide 0.2 g litre1 and 2x methanol. The mobile phase was titrated with solid NaOH to pH 3, followed by titration with CH3COONa to a final pH of 3.6. The flow rate was 1 ml min1. The system was equipped with two detectors in series. Salicylate was identified and measured fluorimetrically using a FD-300 model fluorescence detector (Spectrovision, Chelmsford, MA, USA) using excitation and emission wavelengths of 300 and 412 nm respectively. DHBA derivatives were quantified using an electrochemical amperometric detector (Model 4A; Bioanalytical Systems, West Lafayette, IN, USA), with a plastic cell equipped with a glass carbon electrode operated at +0.80 V, using an AgAgCl reference electrode. The signals from the detector were acquired on an EZChrom data acquisition and handling system (EZChrom EliteTM, Scientific Software Inc., San Ramon, CA, USA) and subsequently processed.
Statistical analysis
Haemodynamic data over time within each group were analysed by analysis of variance (ANOVA) with repeated measures on one factor. Differences in haemodynamics between groups were analysed by ANOVA with the Tukey post hoc test. Incidence of ventricular fibrillation was analysed with the KruskalWallis test. Intergroup comparisons for infarct sizes were made with one-way ANOVA, and group differences were detected with Tukey's post hoc test. Differences between the study groups in DHBA levels were assessed by ANOVA with the Tukey post hoc test. Statistical calculations were performed using SPSS 10.0 for Windows software (SPSS, Chicago, IL, USA). Data are expressed as mean (SEM) and significance was assumed for P<0.05.
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Results |
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Mean arterial pressure was lower in the isoflurane group when the volatile anaesthetic was administered. This returned to normal after the washout period, before ischaemia. Afterwards, there was a trend for mean arterial pressure to decrease over time within all groups. Heart rate was higher (not significantly different) in the isoflurane group. There were no significant differences in ratepressure product within the three groups (Table 1).
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Discussion |
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This study confirms that isoflurane mimics ischaemic preconditioning when infarct size is the end-point. Kersten and colleagues2 showed that isoflurane reduced infarct size in dogs, and this beneficial action was found to persist despite its discontinuation before coronary artery occlusion. This favourable action of isoflurane was also demonstrated in a rabbit model similar to ours.3 The timing of isoflurane administration seems to play an important role. In the study of Preckel and colleagues,12 isoflurane did not affect infarct size in the rabbit in vivo, when it was introduced to the inspired gas a few minutes before reperfusion and was continued for the first 15 min of reperfusion. However, when it was administered and washed out for 1530 min before ischaemia, its effect was comparable to that of ischaemic preconditioning.2 3 9 13 14
The protecting effect of isoflurane on the heart is thought to be mediated by the same end effector as classic preconditioning since there is no additional effect of isoflurane and ischaemic preconditioning when they are administered alone or simultaneously.15 Zaugg and colleagues15 showed in a simulated cellular model of ischaemia that isoflurane mediated its protection in cardiomyocytes by selectively priming mitochondrial KATP channels through multiple triggered protein kinase C-coupled signalling pathways.
The release of reactive oxygen species (ROS) plays a major role in tissue damage and infarction during reperfusion.16 However, release of minute quantities of ROS has been reported to be an important feature of isoflurane-induced preconditioning. Tanaka and colleagues17 and Müllenheim and colleagues18 reported attenuation of isoflurane-induced reduction in myocardial infarct size by the use of ROS scavengers in rabbits.
The oxygen free radicals, including superoxide, are released when neutrophils adhere to the vascular endothelium.5 This production results from activation and assembly of NADH oxidase, which is a transmembrane electron transport chain enzyme that reduces oxygen to superoxide.19 Hanley and colleagues20 have investigated the effects of volatile anaesthetics (halothane, isoflurane and sevoflurane) on electron transport chain activity in intact ventricular myocytes of the guinea-pig. All three volatile anaesthetics inhibited NADH oxidation in submitochondrial particles. However, only halothane was found to inhibit succinate oxidation, suggesting that only halothane blocked complexes II, III and IV of the electron transport chain. In addition, in experiments performed on neutrophil suspensions and coronary rings from mongrel dogs, Hu and colleagues19 found that isoflurane inhibited superoxide production by neutrophils, neutrophil adherence to the endothelial surface of coronary artery segments, and neutrophil-induced coronary endothelial dysfunction, and that these effects could be caused by a direct inhibitory effect on NADH oxidase. The mechanism of isoflurane-decreased superoxide production is not yet known. There are KATP channels that modulate neutrophil activity.20 However, the administration of glibenclamide, a KATP channel blocker, did not alter the ability of isoflurane to inhibit neutrophil superoxide production, suggesting that another mechanism is implicated in this effect. Superoxide is at the origin of other ROS, such as hydroxyl radicals. In the present study, classical ischaemic preconditioning but not isoflurane decreased hydroxyl production during reperfusion. This is in contrast to the work of Nakamura and colleagues,21 who showed that isoflurane and halothane, but not sevoflurane, reduced hydroxyl radical production in the reperfused working rat heart. We have also demonstrated that halothane prevents hydroxyl radical production in an in vivo canine model of ischaemiareperfusion.7 These discrepancies may be attributed to the different models (different species and different protocols) used, implying that different mechanisms of cardioprotection are involved. Hydroxyl radicals are associated with intracellular Ca2+ overload during ischaemia and reperfusion.22 Reduced cytosolic Ca2+ overload caused by increased sarcolemmal KATP channel activity23 (which is one of the pathways of ischaemic preconditioning) has been proposed as a protective mechanism for volatile anaesthetics. Isoflurane decreases this activity in rabbit ventricular myocytes; however, it also decreases the ATP sensitivity of the channel, thus increasing the possibility of opening for a certain ATP concentration.15
In our previous work, we hypothesized that attenuation of free radical production by halothane might be mediated via inhibition of intracellular Ca2+ influx.7 The present findings concerning isoflurane may be explained by the different modes of action of these two volatile anaesthetics on the voltage-sensitive calcium channel. It has already been demonstrated that halothane and enflurane, but not isoflurane, act on a calcium release channel of the sarcoplasmic reticulum.24 Isoflurane only decreases trans-sarcolemmal calcium entry.25
Iron and copper play major roles in biological systems, catalysing free radical production. Hydroxyl radicals are formed from superoxide by Fe-catalysed reactions (the HaberWeiss and Fenton reactions).26 We have shown in isolated rat hearts that ischaemia-induced myocardial iron mobilization was attenuated by ischaemic preconditioning, and that the signalling for ferritin accumulation, a protective step which prepares the heart for accommodating the high load of free iron, was also promoted by ischaemic preconditioning.27 Isoflurane does not seem to have any effect on this mechanism.
The difference in the effects of ischaemic preconditioning and isoflurane on hydroxyl free radical production may indicate that different mechanisms are involved. These mechanisms have yet to be described.
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Acknowledgments |
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References |
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