Cardioprotective effects of desflurane: effect of timing and duration of administration in rat myocardium

Abstract

Background. We compared the cardioprotective effects of 1 minimum alveolar concentration (MAC) desflurane administered before, during or after ischaemia, or throughout the experiment (before, during and after ischaemia) on myocardial infarct size following 30 min occlusion of the left anterior descending coronary artery and 3 h reperfusion in adult rats.

Methods. Fifty male Sprague–Dawley rats were anaesthetized with pentobarbital, intubated and mechanically ventilated. Blood gases, pH and body temperature (37.5–38°C) were controlled. Heart rate and arterial pressure were measured continuously. Animals were randomly assigned to the following groups (n=10 in each group): pentobarbital only (‘Pento’); 15 min desflurane administration followed by 10 min of washout before 30 min ischaemia and 3 h reperfusion (‘Precond’); 30 min desflurane administration during ischaemia period (‘Isch’); desflurane administration during the 15 first min of reperfusion (‘Reperf’) and desflurane administration throughout the experiment (before, during and after ischaemia; ‘Long’). Volumes at risk and infarct sizes were assessed by Indian ink and with 2,3,5‐triphenyltetrazolium chloride staining, respectively.

Results. Physiological parameters and volumes at risk were not significantly different between groups. In the Pento group, mean myocardial infarct size was 65 (sd 15)% of the volume at risk; myocardial infarct size was reduced to a significant and comparable extent in the desflurane‐treated groups (Precond 42 (14)%; Isch 34 (11)%; Reperf 41 (15)%; Long 33 (10)%; P<0.0002 vs Pento group).

Conclusions. In rats, desflurane 1 MAC significantly decreased myocardial infarct size whatever the period and duration of administration.

Br J Anaesth 2004; 92: 552–7

Accepted for publication: November 5, 2003

There is increasing evidence to indicate that volatile anaesthetics in vivo may decrease reversible myocardial ischaemic injury in various species either by pharmacological preconditioning1^–4 or by a protective effect during the reperfusion period.56 Although anaesthetic preconditioning has been widely studied, the reported cardioprotective effect of anaesthesia administered during reperfusion has received less attention. ATP‐dependent potassium channels (K_ATP), protein kinase C and reactive oxygen species have been shown to play a critical role in volatile‐anaesthetic‐induced preconditioning.7^–9 On the other hand, the mechanisms involved in anaesthetic‐induced decrease in reperfusion injury remain unresolved. Finally, the timing and duration of administration of volatile anaesthetics required to induce a cardioprotective effect remain largely unknown. Recently, Varadarajan and colleagues10 suggested that sevoflurane‐induced cardioprotection against global cardiac ischaemia was more effective when sevoflurane was administered before ischaemia than when it was administered only during reperfusion.

In patients, several clinical studies support the observation that volatile‐anaesthetic‐induced myocardial preconditioning may be beneficial during coronary surgery with cardiopulmonary bypass. A 10 min administration of sevoflurane has been shown to precondition the myocardium in patients scheduled for coronary artery bypass graft surgery, resulting in better preservation of myocardial and renal function.11 However, myocardial ischaemia reperfusion may also occur during surgery in high‐risk patients undergoing non‐cardiac surgery. Interestingly, De Hert and colleagues12 reported that sevoflurane‐based anaesthesia may improve myocardial function when compared with propofol in coronary surgery patients, an observation that would suggest that the cardioprotective effect of sevoflurane may be independent of the timing and duration of administration. Thus, it is of particular interest to determine to what extent differential timing and duration of administration of desflurane might induce a cardioprotective effect against ischaemia‐reperfusion injury. The aim of this study was therefore to measure the decrease in myocardial infarct size induced by 1 minimum alveolar concentration (MAC) desflurane at different periods and durations. Desflurane was chosen for this study because it is currently used in clinical practice and because it strongly protects the myocardium in different species in both in vivo and invitro models.4513^–18

Materials and methods

Experimental procedures conformed to the guidelines proposed in the Guide for the Care and Use of Laboratory Animals, and to the appropriate European directives and French national legislation. Fifty adult male Sprague–Dawley rats weighing 280 (50) (mean (sd)) g (R. Janvier Breeding Centre, Le Genest‐St‐Isle, France) were used in the present study. The animals had free access to food and water in an animal room at a constant temperature (22 (1)°C) and humidity (55 (10)%), with a reversed light cycle.

Animal preparation

The rats were anaesthetized with a single i.p. injection of pentobarbital 60 mg kg^–1. No neuromuscular blocking agent was used at any time. After tracheal intubation, the rats were artificially ventilated (Harvard Apparatus 683, Massachusetts, USA) with oxygen 100%. A heparinized catheter (10 UI ml^–1) was inserted into the right femoral artery for continuous monitoring of heart rate and diastolic, mean and systolic arterial pressures and for blood sampling (before, during and after ischaemia) for the analysis of blood gases and pH (Ciba Corning M328, Essex, UK). In all groups, desflurane 5.7%, corresponding to 1 MAC in normothermic rats,19 was delivered in oxygen 100% at different periods according to group. Animals were maintained normothermic (37.5–38°C) during the entire experiment using a feedback‐controlled heating blanket (Harvard Apparatus Ltd, Edenbridge, UK) connected to a rectal probe.

Experimental protocol

The experimental protocol is summarized in Figure 1. After a left thoracotomy and pericardiotomy, the heart was exposed and a suture was passed around the left anterior descending (LAD) coronary artery by inserting a small curved Prolene 6.0 needle into the margin of the pulmonary cone, exiting through the middle of a line linking the cone to the atrium. The suture ends were threaded through a small vinyl tube to prepare a snare. After the surgical procedure was completed (within 45 min), a 10 min stabilization period was allowed and rats were randomly assigned into the five experimental groups described below. In all groups, the LAD coronary artery was occluded for 30 min by tightening the snare. Myocardial ischaemia was confirmed by the appearance of a regional cyanosis on the epicardium distal to the snare and akinesia or bulging in this area. After 30 min ischaemia, the snare was released; the thread passed around the LAD coronary artery was left in place and the wounds were sutured. During the period of reperfusion (3 h), the rats progressively awakened and were allowed to move freely in the cage. At the end of the reperfusion period, the rats were re‐anaesthetized with pentobarbital 30 mg kg^–1 for about 15 min. The coronary artery was briefly re‐occluded and diluted Indian ink was injected into the femoral artery catheter to delineate the area at risk in vivo. With this technique, the previously non‐ischaemic area appeared in black whereas the area at risk remained unstained. Under anaesthesia, the heart was excised, frozen and cut into eight or nine 1 mm thick transverse slices. Each slice was then incubated for 45 min in a solution of 2,3,5‐triphenyltetrazolium chloride 1% (Sigma, Saint Quentin Fallavier, France) to differentiate infarcted area (pale) from viable (red) myocardial area and the slices were digitized.20 The extent of left ventricular area, area at risk and infarcted regions were delineated using the public‐domain ImageJ software. Volumes were calculated by integrating areas over all slices. Infarct sizes were calculated as percentages of volume at risk.

The groups were as follows (see also Fig. 1). ‘Pento’ (n=10): single injection of pentobarbital 60 mg kg^–1 before the surgical preparation, no pentobarbital or desflurane administration during the entire experiment; animals were awake 30 min after reperfusion; ‘Precond’ (n=10): single injection of pentobarbital 60 mg kg^–1 before the surgical preparation, 15 min administration of desflurane 1 MAC followed by a 10 min washout; there was no administration of desflurane during ischaemia and reperfusion periods; animals were awake 30 min after reperfusion; ‘Isch’ (n=10): single injection of pentobarbital 60 mg kg^–1 before the surgical preparation, 30 min administration of desflurane 1 MAC during the ischaemia period; during preconditioning, washout, and reperfusion periods, there was no administration of desflurane; animals were awake 30 min after reperfusion; ‘Reperf’ (n=10): single injection of pentobarbital 60 mg kg^–1 before the surgical preparation, 15 min administration of desflurane 1 MAC during the first 15 min of the reperfusion period; during, preconditioning, washout and ischaemia periods, there was no administration of desflurane; animals were awake 30 min after reperfusion; ‘Long’ (n=10): single injection of pentobarbital 60 mg kg^–1 before the surgical preparation, administration of desflurane anaesthesia 1 MAC was maintained for 1 h 10 min, from the beginning of the preconditioning period until the end of the first 15 min of reperfusion; animals were awake 1 h after reperfusion.

Statistical analysis

Values are given as mean (sd). Data were analysed using Statview^® software (Abacus Concepts, Berkeley, USA). Left ventricular weights, volumes at risk and infarct sizes were compared by anova, followed by a Student’s t‐test with Bonferroni correction.

For each phase of the experiment (baseline, preconditioning, washout, beginning of ischaemia (first 15 min), end of ischaemia (last 15 min), reperfusion) average values for rectal temperature, diastolic, mean and systolic arterial pressures and cardiac frequency were calculated for each animal.

Physiological parameters were analysed by a two‐way repeated‐measures anova (factors: group, time as repeated factor), followed by Student’s t‐tests with Bonferroni correction. P<0.05 was accepted as significant.

Results

Myocardial infarct sizes

Infarct sizes in the five groups are summarized in Table 1 and Figure 2. No significant differences between the groups were observed for the volume at risk and left ventricular weight. Infarct sizes were calculated as percentages of volume at risk. In all groups treated with desflurane, a significant reduction of infarct size (P<0.0002) was obtained compared with the Pento group. No significant difference was observed between the desflurane‐treated groups.

Physiological parameters

All physiological parameters were within the physiological range (Pa_O2 mean 36.4 (sd 12) kPa, Pa_CO2 4.91 (0.46) kPa, pH 7.42 (0.04), temperature mean 37.7 (sd 0.3)°C). For Pa_CO2, Pa_O2, pH and temperature, there was no interaction between group and time (P>0.063). There were no significant differences between the five groups at any time.

Mean arterial pressure and heart rate are summarized in Table 2. For diastolic, mean and systolic arterial pressure, there were significant interactions between group and time (P<0.0001). There was a significant difference between the Isch and Reperf groups during the first 15 min of ischaemia for diastolic pressure (P<0.03). All other comparisons did not reveal any significant differences.

For heart rate, there was a significant interaction (P<0.012) in a two‐way anova. There was a significant difference between the Reperf and Precond groups as compared with the Long group during the first 15 min of the ischaemic period (P<0.0001). All other comparisons were not significantly different.

Discussion

The present study shows that desflurane 1 MAC induced a significant reduction in myocardial infarct size irrespective of the period and duration of administration. Our present study thus confirms and extends the results of previous investigations showing that desflurane preconditions the myocardium against ischaemia and decreases reperfusion injury.

It could be argued that the anaesthetic protocol using pentobarbital might have influenced our results. The literature concerning a potential cardioprotective effect of pentobarbital is equivocal. Pentobarbital reduces myocardial infarction after 90 min of ischaemia in dogs.21 Another study shows that infarcts were larger in dogs treated with pentobarbital as compared with a conscious dog model.22 Moreover, Cope and colleagues1 have shown that pentobarbital does not increase the extent of myocardial infarction in the rabbit myocardium. In our study, all groups received the same anaesthetic protocol and thus it is unlikely that the barbiturate could have differentially influenced the cardioprotective effect measured in the desflurane‐treated groups. Therefore, the decrease in myocardial infarct size in the desflurane‐treated groups could be attributed to desflurane itself.

It should be highlighted that all the physiological parameters measured were not different for the group factor. Although we have observed differences in arterial pressure and heart rate as a factor of time in each group when desflurane was administered, these differences remained modest and without any biological significance. Furthermore, Cope and colleagues1 have previously shown a lack of correlation between infarct size and the hypotensive effects of volatile anaesthetics.

Myocardial desflurane‐induced preconditioning has been well established in various experimental models and species. Thirty min desflurane 1 MAC administration before coronary occlusion has been shown to decrease infarct size in dogs in vivo.18 Furthermore, Piriou and colleagues4 have shown that desflurane 1 MAC may result in a more potent cardioprotective effect than the other volatile anesthethics. Importantly, Hanouz and colleagues13 have shown that a 10 min administration of 3%, 6% and 9% desflurane preconditions human atrial myocardium against 30 min of simulated ischaemia in vitro. Our study also confirms that a short period of administration of desflurane before ischaemia reduces myocardial infarct size from 65% to 42% of the area at risk. Furthermore, the degree of protection obtained in the present work is similar to that found previously for desflurane preconditioning.4

Desflurane‐induced preconditioning may result from activation of several signalling pathways that remain incompletely understood. The opening of mitochondrial and sarcolemmal K_ATP channels is considered as a major step in the preconditioning cascade.7 In dogs, both sarcolemmal and mitochondrial K_ATP channels could intervene in in vivo desflurane‐induced cardioprotection.18 In contrast, in the human myocardium, sarcolemmal K_ATP channels seem to play a less prominent role.13 In addition, numerous triggering stimuli may be involved in desflurane‐induced myocardial preconditioning, such as adenosine A₁ receptor and α‐ and β‐adrenoceptor stimulation.13 Finally, protein kinase C and reactive oxygen species have been shown to play a pivotal role in volatile‐anaesthetic‐induced preconditioning.189

Although recent studies suggest that myocardial preconditioning induced by volatile anaesthetics may be of benefit in some patients, it remains limited to cardiac surgery with cardiopulmonary bypass. However, myocardial ischaemia may occur during scheduled or emergency surgery in high‐risk patients. Maintenance of general anaesthesia in high‐risk patients may include volatile anaesthetics, especially desflurane or sevoflurane, which better preserve haemodynamic status. In such situations, myocardial ischaemia could not be anticipated and may occur when anaesthesia is maintained, at least in part, with volatile anaesthetics. On the other hand, transient myocardial ischaemia may occur before surgery and then volatile anaesthetics will be administered during the reperfusion period.

Desflurane has been shown to reduce myocardial injury and to improve contractile function during the reperfusion period. In the rabbit heart in vivo, when given solely during the reperfusion period, desflurane 1 MAC limited myocardial necrosis.5 Schlack and colleagues14 have shown that functional recovery was improved by volatile anaesthetics (1.5 MAC) administered during the first 30 min of reperfusion, and that the recovery induced by desflurane occurred earlier than with other volatile anaesthetics. Furthermore, desflurane 1.5 MAC inhibited spontaneous postinfarction ventricular dysrhythmias when administered after 22 h of infarction in dogs.15 Our results showing that desflurane 1 MAC reduces infarct size when administered during the first 15 min of the reperfusion period are in accordance with previous studies. Furthermore, the degree of protection obtained in the present work is similar to that found earlier for desflurane administered only during the reperfusion period.5

The mechanims of volatile‐anaesthetic‐induced decreased reperfusion injury remain unresolved. Nevertheless, beneficial effects on intracellular calcium homeostasis,23 leucocyte and platelet activation and adhesion, and on release of oxygen‐derived free radicals24 have been shown to be involved. Finally, it has been shown recently that cardioprotective pathways could be activated during the reperfusion period, and that this postconditioning phenomenon was as effective as preconditoning in decreasing myocardial infarct size.25 Horn and colleagues26 have shown that desflurane reduces the aggregation between platelets and leucocytes, neutrophils and monocytes. This latter study suggests that desflurane can protect the heart during the ischaemia–reperfusion sequence.

Our study shows for the first time that desflurane‐induced decrease in infarct size persists if the anaesthetic is administered only during the ischaemic period. Pagel and colleagues16 reported that desflurane administered during ischaemia attenuates myocardial stunning after 10 min of LAD coronary artery occlusion in chronically instrumented dogs. Another study also shows that 1.1 and 1.6 MAC desflurane exert beneficial effects on diastolic function in chronically instrumented dogs in the presence of transient myocardial ischaemia.17 Our study confirms and extends this result showing that desflurane administered during transient ischaemia decreases myocardial infarct size following reperfusion. Moreover, the decrease in myocardial infarct size is comparable to that observed in the desflurane‐preconditioned group. These observations suggest that cardioprotective signalling pathways may also be activated during the ischaemic period. However, the precise mechanisms involved in the desflurane‐induced cardioprotection observed during the ischaemic period remain unknown and require further studies.

Most interestingly, our study also shows that desflurane decreases myocardial infarct size following transient ischaemia occurring during desflurane administration. The cardioprotective effect of desflurane measured in the Long group is comparable with that measured in the Precond and Reperf groups (Fig. 2). This result is of particular importance because it suggests that desflurane‐induced cardioprotective effects may also be effective whatever the timing and duration of administration. In clinical practice, it has been shown that sevoflurane‐based anaesthesia better preserves left ventricular function after cardiopulmonary bypass when compared with propofol‐based anaesthesia.12 In addition, this study of De Hert and colleagues12 shows less evidence of myocardial damage in the first 36 h after surgery in a group of patients subjected to sevoflurane anaesthesia. The mechanisms involved during desflurane‐based anaesthesia remain unknown. It could be hypothesized that multiple signalling pathways involved in desflurane‐induced preconditioning may be also activated.

In conclusion, under our experimental conditions, we have shown that desflurane affords an effective protection against transient focal cardiac ischaemia in rats. This protection is independent of the period of desflurane administration.

Acknowledgements

These investigations were supported by grants from the CNRS and the University of Caen. We thank Professor C. Thuillez and his group (INSERM EA9212) for the demonstration of the focal ischaemia surgery in the heart.

Fig 1 Experimental protocol. The preparation of each animal lasted approximately 45 min. Occlusion of the left anterior descending (LAD) coronary artery was maintained for 30 min, followed by reperfusion. After 3 h, areas at risk and infarcts were revealed by Indian ink infusion and 2,3,5‐triphenyltetrazolium chloride staining, respectively. Five groups were studied which differed according to the period and the duration of desflurane administration, as shown. Desflurane 5.7% was delivered in oxygen 100%. BL, baseline period.

Fig 2 Infarct size (% volume at risk) in the five groups. Pento, pentobarbital group; Precond, desflurane administered during the preconditioning period; Isch, desflurane administered during ischaemia; Reperf, desflurane administered during the first 15 min of reperfusion; Long, desflurane administered during the entire sequence (preconditioning, washout, ischaemia, reperfusion). Average values are indicated as means and sd to the right of individual values. *Significant difference between the pentobarbital‐treated and all other groups (P<0.0001; t‐tests with Bonferroni correction following anova).

Table 1

Left ventricular weight, volume at risk and infarct size for each group (n=10). Pento, pentobarbital group; Precond, desflurane administered during the preconditioning period; Isch, desflurane administered during ischaemia; Reperf, desflurane administered during the first 15 min of reperfusion; Long, desflurane administered during the entire sequence (preconditioning, washout, ischaemia, reperfusion). Values are mean (sd) analysed by anova (see text for details). *P<0.0002 vs Pento group

	Pento	Precond	Isch	Reperf	Long
Left ventricular weight (mg)	804 (113)	751 (78)	803 (101)	804 (103)	890 (107)
Volume at risk (% left ventricle)	40 (11)	38 (12)	30 (10)	31 (8)	32 (12)
Infarcted risk zone (% volume at risk)	65 (15)	42 (14)*	34 (11)*	41 (15)*	33 (10)*

Table 2

Heart rate (HR) and mean arterial pressure (MAP) for the five groups during baseline, ischaemia and the first 15 min of reperfusion. Pento, pentobarbital group; Precond, desflurane administered during the preconditioning period; Isch, desflurane administered during ischaemia; Reperf, desflurane administered during the first 15 min of reperfusion; Long, desflurane administered during the entire sequence (preconditioning, washout, ischaemia, reperfusion). Values are mean (sd) analysed by anova (see text for details). *P<0.0001 vs Long group

	Baseline		Ischaemia		Reperfusion (first 15 min)
	HR	MAP	HR	MAP	HR	MAP
Pento	397 (66)	121 (25)	400 (56)	114 (21)	382 (96)	111 (19)
Precond	432 (57)	112 (12)	448 (46)*	110 (11)	410 (53)	99 (17)
Isch	447 (46)	118 (14)	413 (54)	96 (11)	404 (54)	111 (11)
Reperf	454 (48)	120 (6)	447 (37)*	116 (9)	359 (88)	95 (14)
Long	408 (53)	128 (17)	373 (43)	109 (25)	347 (28)	102 (30)

Author notes

1University of Caen: UPRES EA 3212, IFR47; Département d‘Anesthésie Réanimation, Centre Hospitalier Universitaire (CHU), Côte de Nacre, Caen, France. 2University of Caen, CNRS: UMR‐6551, IFR47; CYCERON Centre, Boulevard Henri Becquerel, BP 5229, F‐14074 Caen Cedex, France