Pharmacological preconditioning: comparison of desflurane, sevoflurane, isoflurane and halothane in rabbit myocardium{dagger}

V. Piriou*,1, P. Chiari1, F. Lhuillier1, O. Bastien1, J. Loufoua2, O. Raisky1, J. S. David1, M. Ovize2 and J.-J. Lehot1

1 Service d’Anesthésie Réanimation and 2 Laboratoire de physiologie Lyon Nord, Hôpital cardio-vasculaire Louis Pradel, Avenue Doyen Lépine, F-69500 Lyon Bron, France *Corresponding author

{dagger}This work was presented at the 23rd annual meeting of the Society of Cardiovascular Anesthesiologists, Vancouver, Canada, May 2001 (Anesth Analg 2001; 92: SCA39).

Accepted for publication: April 30, 2002


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Background. Recent investigations showed that isoflurane can induce pharmacological preconditioning. The present study aimed to compare the potency of four different halogenated anaesthetics to induce preconditioning.

Methods. Anaesthetized open-chest rabbits underwent 30 min of coronary artery occlusion followed by 3 h of reperfusion. Before this, rabbits were randomized into one of five groups and underwent a treatment period consisting of either no intervention for 45 min (control; n=10), or 30 min of 1 MAC halogenated anaesthetic inhalation followed by 15 min of washout. End-tidal concentrations of halogenated agents were 3.7% for sevoflurane (n=11), 1.4% for halothane (n=9), 2.0% for isoflurane (n=11), and 8.9% for desflurane (n=11). Area at risk and infarct size were assessed by blue dye injection and tetrazolium chloride staining.

Results. Mean (SD) infarct size was 54 (18)% of the risk area in untreated controls and 40 (18)% in the sevoflurane group (P>0.05, ns). In contrast, mean infarct size was significantly smaller in the halothane, isoflurane, and desflurane groups: 26 (18)%, 32 (18)% and 16 (17)%, respectively (P<0.05 vs control).

Conclusions. Halothane, isoflurane and desflurane induced pharmacological preconditioning, whereas sevoflurane had no significant effect. In this preparation, desflurane was the most effective agent at preconditioning the myocardium against ischaemia.

Br J Anaesth 2002; 89: 486–91

Keywords: anaesthetics volatile; heart, myocardial function


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
There is growing evidence that halogenated anaesthetics are cardioprotective. They decrease infarct size following a prolonged ischaemic insult13 and improve postischaemic functional recovery (i.e. myocardial stunning).46 Preconditioning is an endogenous phenomenon whereby repeated brief episodes of coronary artery occlusion protect the heart against further sustained ischaemia.7 8 Recent studies have demonstrated that administration of some pharmacological agents, including halogenated anaesthetics, before prolonged ischaemia can precondition the heart (‘pharmacological preconditioning’).9 10 In 1997, Cope and colleagues,9 using a Langendorff rabbit model, Kersten and colleagues10 and Cason and colleagues,11 using dog and rabbit in vivo models, reported that the pre-administration of isoflurane followed by a washout period preconditioned the heart. Later studies have used isoflurane to investigate the underlying mechanisms of this protection.1214 Activation of mitochondrial KATP channels is probably involved in isoflurane-induced preconditioning.14 Halothane has been shown to induce pharmacological preconditioning in an in vitro model.9 Four different inhalational anaesthetic agents are currently used in our clinical practice: halothane, isoflurane, sevoflurane and desflurane. However, whether their myocardial preconditioning potency is equivalent is unknown. The aim of the present study was therefore to compare the efficacy of these four halogenated agents at triggering pharmacological preconditioning in the in vivo rabbit model.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
All experiments performed in this study conformed to the Guiding Principles in the Care and Use of Animals approved by the American Physiologic Society. The Laboratoire de Physiologie Lyon Nord and the investigators had authorization from the French government to perform such animal studies.

A total of 73 New Zealand White rabbits of either sex (2.0–2.5 kg) were premedicated with xylazine 5 mg kg–1 i.m. and anaesthetized with ketamine hydrochloride 50 mg kg–1 i.m. Anaesthesia was maintained by continuous infusion of a mixture of ketamine 3 mg kg–1 h–1 and xylazine 1.5 mg kg–1 h–1. Adequate depth of anaesthesia was ensured before any surgical procedures by the absence of pedal and palpebral reflexes. Following tracheostomy, the animal’s lungs were ventilated mechanically (Servo ventilator 900B, Siemens-Elema, Sweden and SA2 ventilator, Dräger, Lübeck, Germany) with 100% oxygen. Tidal volume was set at 15 ml kg–1 and the respiratory rate to 35 bpm. Ventilation was adjusted to maintain end-tidal CO2 E'CO2 in the physiological range. End-tidal gas concentrations were measured continuously (gas analyser Capnomac Ultima, Datex, Helsinki, Finland). Body temperature, recorded through a thermistor inserted into the oesophagus, was maintained between 39.0 and 40.5°C by means of a servo-controlled heating element incorporated into the operating table. Limb lead II of the ECG was monitored continuously by means of subcutaneous needle electrodes. Systemic arterial pressure was monitored using a Gould pressure transducer connected to a 1-mm fluid-filled catheter inserted in the right carotid artery. The right internal jugular vein was catheterized with a 1 mm catheter to infuse fluids and drugs. Hetastarch 5 ml kg–1 h–1 was infused continuously via this intravenous cannula. Fentanyl 50 µg i.v. was injected before thoracotomy to provide adequate analgesia.

The heart was exposed via a left thoracotomy. The first large marginal branch of the circumflex artery was identified and a 5/0 Dexon suture was passed around this artery, approximately halfway between the apex and the base. The suture ends were threaded through a small vinyl tube to make a snare to perform further coronary occlusion and reperfusion. After the surgical procedure, a 15-min stabilization period was allowed.

In all groups, the coronary artery was occluded for 30 min. Myocardial ischaemia was confirmed by the appearance of a regional cyanosis on the epicardium distal to the snare, akinesia or bulging in this area, and a marked ST segment elevation on the ECG. After 30 min, the snare was released and reperfusion allowed for a period of 3 h. Reperfusion was visually confirmed by the appearance of hyperaemia. The thread passed around the marginal artery was left in place.

At the end of the reperfusion period, the coronary artery was briefly re-occluded and diluted Uniperse blue (Ciba-Geigy, Hawthorne, NY, USA) was injected into the jugular vein to delineate the in vivo area at risk. With this technique, the previously non-ischaemic area appears blue whereas the area at risk remains unstained. Anaesthetized rabbits were then injected with potassium chloride (1 g) and the heart was excised and cut into five or six 2-mm-thick transverse slices.

After removing right ventricular tissue, each slice was weighed and identified. The basal surface of each slice was photographed for future measurement of the area at risk. Each slice was then incubated for 15 min in tetrazolium chloride to differentiate infarcted (pale) from viable (red) myocardial area.15 Each slice was then photographed again. Using enlarged projections, the boundaries of the different areas on each slice were traced. The extent of left ventricle (LV) area, area at risk and infarct size were quantified by computerized planimetry (ImageJ software, version 1.01z, National Institutes of Health, USA) and corrected for the weight of the tissue slice. Total weights of area at risk and area of necrosis were then calculated and expressed as weight (g) or as percentages of total LV weight. It was decided prospectively that hearts with a risk region <10% of the LV weight would be excluded from the study.

Experimental groups
Animals were randomly assigned into five groups (Fig. 1). All groups underwent a 30-min coronary artery occlusion and 3 h of reperfusion. Before this prolonged ischaemia, they underwent a 45-min treatment period. After the 15-min stabilization period, the halogenated anaesthetic was added to the inspired gas for 30 min and discontinued 15 min before ischaemia. Volatile anaesthetics were titrated to an end-tidal concentration of 3.7% sevoflurane, 1.4% halothane, 2.0% isoflurane and 8.9% desflurane, corresponding to 1.0 MAC of each anaesthetic in the rabbit.1618 For each animal, end-tidal concentration of halogenated agent was less than 0.1% at the end of the washout period.



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Fig 1 Experimental protocol. Filled bars represent ischaemia; empty bars represent perfusion. Sevoflurane, halothane, isoflurane and desflurane were titrated to end-tidal concentrations of 3.7%, 1.4%, 2.0% and 8.9%, respectively.

 
Isoflurane and sevoflurane were purchased from Abbott laboratories (Queenborough, UK), halothane from Zeneca Pharma (Cergy, France) and desflurane from Baxter (Lessines, Belgium).

Statistics
Statistical analysis of haemodynamics and E'CO2 was performed using two-way ANOVA with repeated measures on one factor. LV weight and area at risk were analysed by analysis of variance. Effect of pretreatment on percent of risk zone infarcted was analysed by one-way analysis of variance followed by Dunnett’s post test when appropriate. Differences in infarct sizes among groups were evaluated by analysis of covariance and post-hoc least significance difference test, with infarct size as the dependent variable and area at risk as the covariant. Statistical calculations were performed using Statistica 5.0 (Statsoft Inc., Tulsa, OK, USA) and GraphPad InStat version 3.00 for Windows 95 (GraphPad Software, San Diego, CA, USA). All values are expressed as mean (SD). P<0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Mortality and exclusion
Of the 73 rabbits included in this study, seven were excluded because of fatal ventricular fibrillation during coronary occlusion (one control, one sevoflurane, one halothane, two isoflurane and two desflurane) and six because of severe heart failure (obvious cardiac dilatation and systolic arterial pressure <40 mm Hg) during coronary occlusion or reperfusion (one control, two sevoflurane, one halothane, one isoflurane and one desflurane). Seven rabbits were excluded because of technical problems during surgical preparation and one because the photograph of the heart slices was unreadable. Data are presented for the 52 remaining rabbits: 10 control and 11 sevoflurane-, 9 halothane-, 11 isoflurane- and 11 desflurane-treated rabbits.

Haemodynamic data
Haemodynamic data, including heart rate and arterial pressure, are summarized in Table 1. Inhalational anaesthetic administration decreased systolic arterial pressure consistently but transiently in all groups. This effect was statistically significant for the sevoflurane and desflurane groups. E'CO2 and body temperature did not differ between the groups.


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Table 1 Haemodynamic measurements in different experimental groups. Data are mean (SD). *P<0.05 vs control
 
Infarct size
Data for the area at risk and infarct size are presented in Table 2 and Figure 2. LV weight and area at risk were comparable between the different groups, with mean value of area at risk ranging from 30 (10)% to 47 (13)% of LV weight.


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Table 2 Area at risk, infarct size and risk zone infarcted. Data are mean (SD). LV, left ventricle. *P<0.05 vs control
 


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Fig 2 Infarct size expressed as percentage of area at risk for each individual animal. Horizontal lines are means. Halothane, isoflurane and desflurane pre-administration significantly reduced infarct size; sevoflurane pre-administration had no effect. *P<0.05 vs control.

 
Infarct size in the control group was 54 (18)% of the area at risk. Pretreatment with sevoflurane failed to significantly decrease infarct size (40 (18)% of area at risk) whereas pre-administration of halothane, isoflurane or desflurane significantly attenuated myocardial infarct size (26 (18)%, 32 (18)% and 16 (17)% of area at risk, respectively).

These data were confirmed when comparing the weights of the infarct size with area at risk as a covariant, which represents a major determinant of myocardial infarction in the rabbit model (Table 2).


    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The present study showed that a 30-min pre-administration of halothane, isoflurane or desflurane, followed by a 15-min washout period, significantly decreased myocardial infarct size following a subsequent prolonged ischaemic insult. Desflurane was the most potent agent. Sevoflurane had no significant effect in this model.

Several studies have reported the cardioprotective effects of halogenated anaesthetics against ischaemia-reperfusion, depending on the timing of administration and the ischaemic time. Warltier and colleagues4 and Meissner and colleagues5 showed that halothane, isoflurane and desflurane can attenuate myocardial stunning. All four inhalational anaesthetics can limit infarct size in in vivo preparations, whether they have been administered during a sustained ischaemia or during the reperfusion. When given during a sustained ischaemic period, halothane1 and isoflurane2 decreased infarct size. When given only during the reperfusion period, desflurane and sevoflurane limited myocardial necrosis, whereas isoflurane had no effect.19 A 30-min inhalation of sevoflurane3 or desflurane20 discontinued just before the onset of ischaemia also had significant anti-necrotic effect.

Pre-administration of isoflurane also exerted a protective effect by reducing infarct size when the discontinuation of this volatile agent was followed by a washout period before ischaemia. This isoflurane-induced pharmacological preconditioning has been shown in vitro9 and in vivo.10 11 Similar observations have been made using halothane and enflurane.9 Similar to ischaemic preconditioning, isoflurane-induced preconditioning involves adenosine receptors,12 Gi protein13 and mitochondrial KATP channels.14 Adenosine A1 receptors and protein kinase C can also modulate halothane-induced preconditioning.9

Our study is the first to address the issue of the intensity of the preconditioning effect of these four halogenated agents in the same experimental conditions. We found that the degree of protection differed between the four agents. Sevoflurane failed to significantly reduce infarct size. While halothane and isoflurane appeared very similar, desflurane displayed the most potent cardioprotective effect. Consistent findings have been reported by Toller and colleagues using a canine model.3 The authors showed that discontinuation of a 30-min administration of 1 MAC sevoflurane just before onset of ischaemia decreased infarct size. However, when sevoflurane was discontinued 30 min before ischaemia, the myocardial protective effect disappeared. Two-minute ischaemic preconditioning was not long enough to precondition the heart. However, 2-min ischaemia associated with a 30-min sevoflurane pre-administration and a 30-min washout period decreased infarct size. Preconditioning induced by desflurane has not been shown previously. As with ischaemic preconditioning and isoflurane-induced preconditioning, KATP channels may be responsible for desflurane cardioprotection.20

The reasons for the differences between the four inhalational agents remain unclear. Hypotension induced by volatile agents does not seem to be involved in the volatile-anaesthetic-induced preconditioning, the main mechanism involved being a direct effect on KATP channels.10 In the current study, desflurane and sevoflurane were the most hypotensive agents, with opposite effects on infarct size. This is in agreement with Cope and colleagues,9 who showed a lack of correlation between infarct size and the hypotensive effects of volatile anaesthetics. Although +dp/dt and –dp/dt were not measured, it is possible that the different lusitropic and inotropic effects of the four anaesthetics might influence these results.

The basal anaesthetic protocol might influence our results, because ketamine has been shown to block preconditioning21whereas {delta} opiates such as fentanyl confer cardioprotection by mimicking ischaemic preconditioning.22 However, all groups received the same anaesthetic protocol, and it is unlikely that this could explain the differences between groups.

Like ischaemic preconditioning,23 some volatile anaesthetics, such as halothane and isoflurane, can inhibit apotosis.24 Although we did not measure tissue ATP concentrations or assess apoptosis, we cannot rule out that sevoflurane pre-administration can be protective by decreasing apoptosis without significantly decreasing infarct size.

One possible explanation of the most pronounced effect of desflurane in our study is an increase in sympathetic activity25 or the release of myocardial catecholamines26 induced by this agent. Indeed, it has been shown that {alpha}-adrenergic agonists may trigger ischaemic precon ditioning.27 28 Regarding an increase in sympathetic activity, desflurane-induced hypotension was not concomitant of tachycardia in the present study, which might signify a lack of sympathetic activation. Moreover, Pac-Soo and colleagues29 showed in a rabbit model that although low concentrations of isoflurane (1.2%) and desflurane (6%) increased renal sympathetic nerve activity, higher concentrations (such as those used in our study) depressed sympathetic activity. On the other hand, Gueugniaud and colleagues suggested that desflurane may be responsible for myocardial catecholamine release in isolated rat myocardium.26 However, it is not known how myocardial catecholamine release may influence desflurane-induced preconditioning.

Toller and colleagues3 suggested that the lack of effect of sevoflurane might be explained by the low blood gas solubility coefficient (0.62),30 resulting in less residual drug in the myocardium at the end of the washout period than with isoflurane. This does not fit with our data, however, since we demonstrated that desflurane, with the lowest blood–gas partition coefficient (0.49),30 exhibited the most pronounced effect.

We only examined the effects of 1 MAC; however, we cannot rule out that there is a dose–response curve and that a higher concentration of sevoflurane might be effective in preconditioning the rabbit heart.

In conclusion, our results indicate that halothane, isoflurane and desflurane, but possibly not sevoflurane, can induce pharmacological preconditioning. Further studies are needed to address the selective efficacy and mechanism of action for each of these agents.


    Acknowledgements
 
This study was supported in part by the Department of Anaesthesiology and Intensive Care, Association pour la Recherche Cardio-Vasculaire, Pr JJ Lehot, Hopital cardio-vasculaire et Pneumologique Louis Pradel, Lyon, France and by the Fondation de France, Bourse de recherche Bullukian (Champagne au Mont d’Or, France). A special acknowledgement is due to our laboratory technician, Florence Arnal.


    References
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 Introduction
 Material and methods
 Results
 Discussion
 References
 
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