1 Laboratory of Experimental Anaesthesiology and Cellular Physiology, University of Caen, UPRES EA 3212, Département dAnesthésie Réanimation, Centre Hospitalier Universitaire (CHU), Côte de Nacre, Caen, France. 2 Laboratory of Neuronal Death, Neuroprotection and Neurotransmission, University of Caen, CNRS, UMR-6551, CYCERON Center, Boulevard Henri Becquerel, BP 5229, F-14074 Caen Cedex, France
Corresponding author
Accepted for publication: April 11, 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. Male SpragueDawley rats were anaesthetized with desflurane or halothane, intubated, and mechanically ventilated. Mean arterial pressure (MAP), blood gases, and pH were controlled. Body temperature was maintained at 37.538°C. Animals were assigned to one of four groups according to the anaesthetic type (halothane or desflurane) and the duration of anaesthesia: short-duration, during the preparation only; long-duration, during both preparation and ischaemia. Twenty-four hours after MCAo, infarcts were visualized by staining with 2,3,5-triphenyltetrazolium chloride. Two additional groups of rats were subjected to the same protocol as that of long-duration halothane and long-duration desflurane with additional pericranial temperature measurements made.
Results. Physiological parameters were comparable between the groups but MAP was higher (P<0.0001) in the short-duration groups. In the short-duration groups, cerebral infarct volumes were not significantly different between anaesthetics (short-duration halothane: 288 (61) mm3, mean (SD); short-duration desflurane: 269 (71) mm3, P>0.56). Compared with the awake state (short-duration groups), halothane and desflurane significantly reduced infarct volumes (long-duration halothane: 199 (54) mm3, P<0.0047 vs short-duration halothane; long-duration desflurane: 121 (55) mm3, P<0.0001 vs short-duration desflurane). The mean infarct volume in the long-duration desflurane group was significantly lower than that in the long-duration halothane group (P<0.0053). Pericranial temperatures were similar in the desflurane and halothane long-duration groups (P>0.17).
Conclusions. In rats, desflurane-induced neuroprotection against focal cerebral ischaemia was greater than that conferred by halothane.
Br J Anaesth 2003; 91: 3906
Keywords: anaesthetics volatile, desflurane; anaesthetics volatile, halothane; brain, neuroprotection; complications, middle cerebral artery occlusion; rat
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animal preparation
Animals were randomly selected to be anaesthetized with halothane (n=18) or desflurane (n=18) always delivered in oxygen:nitrous oxide (30:70%). After tracheal intubation, the animals lungs were artificially ventilated (Harvard Apparatus 683, MA, USA). Throughout the preparation period (45 min), halothane and desflurane concentrations were maintained at 1.51.6 and 1112%, respectively, corresponding to 2.0 MAC.6 7 A catheter was inserted into the right femoral artery for continuous monitoring of mean arterial pressure (MAP) and the periodic analysis of blood gases and pH (Ciba Corning M328, Essex, UK). The catheter was tunnelled subcutaneously and exteriorized at the neck for use in awake animals. The animals were maintained normothermic (37.538°C) using a feedback controlled heating blanket (Harvard Apparatus Limited, Edenbridge, UK) connected to a rectal probe.
Experimental protocol and design
MCAo was performed by insertion of an intraluminal filament (made of a terminal cylinder of melting glue, 2 mm long, diameter 0.38 mm, attached to a nylon thread, 0.22 mm in diameter) into the lumen of the right external carotid artery. The filament was advanced into the internal carotid artery, 9 mm after the outer table of the skull and then secured to the external carotid artery.8 In this manner, the terminal cylinder is reproducibly placed at the MCA origin. For each anaesthetic, the rats were then randomly assigned to one of two experimental groups, which differed by the duration of anaesthesia. Four experimental groups were studied (Fig. 1).
|
Long-duration halothane: anaesthesia was maintained with halothane 1.11.2% (1.5 MAC).
Short-duration desflurane: desflurane anaesthesia was discontinued and the trachea was extubated and again the animals were allowed to spontaneously breathe air.
Long-duration desflurane: anaesthesia was maintained with desflurane 89% (1.5 MAC).
During the period of ischaemia, the rats in the short-duration groups were allowed to move freely in the cage. Their body temperature was measured regularly by transiently inserting a rectal probe and was maintained with a heating lamp. After 2 h, these rats were re-anaesthetized for about 10 min with the same anaesthetic as used previously to withdraw the filament. In all rats, the filament was then removed, wounds were sutured and anaesthesia was discontinued.
Twenty-two hours later, the animals were again anaesthetized with the same anaesthetic as used previously and perfused transcardially at 120 mm Hg with heparinized saline for 2 min followed by 2,3,5-triphenyltetrazolium chloride 2% (Sigma, Saint Quentin Fallavier, France) for 8 min. The brain was removed and fixed by immersion in a paraformaldehyde solution 4%. The brain was then cut in 1 mm slices using a purpose made matrix. The slices were digitized. Infarcted regions were delineated using the public domain ImageJ software. The volume of infarction was calculated by integration over the whole brain of the infarcted surfaces.
In additional experiments, pericranial temperature was measured continuously with a probe inserted between the skull and the temporal muscle in rats exposed to the same protocol as that of long-duration halothane (n=5) and long-duration desflurane (n=5) groups.
Statistical analysis
Values are given as mean (SD). Data were analyzed by ANOVA using Statview® software (Abacus Concepts, Berkeley, CA, USA) as detailed in the results section followed when appropriate by a two-tailed Students t-test. P<0.05 or P<0.05/number of comparisons (Bonferroni correction) was accepted as significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
PaO2, PaCO2, arterial pH, and Temp were analysed using a three-way repeated measures ANOVA (factors: anaesthetic, duration of anaesthesia, time as repeated factor). For PaCO2 and Temp, there were no significant interactions and no main effects, except for Temp for which a significant difference (0.2°C) between the short-duration and long-duration groups was obtained (P=0.0003). For PaO2 and pH, interactions with time were found (P<0.03), and data obtained before and after MCAo were subsequently analysed separately. For these parameters, a three-way repeated measures ANOVA was performed on data obtained during ischaemia. No interaction (P>0.26) with time was found so that values obtained after MCAo were averaged.
Next, two-way factorial ANOVA (anaesthetic, duration) was performed before and after MCAo. All data and statistics are summarized in Table 1. For PaO2 and pH, a significant effect of the duration factor on pH was obtained (P<0.02). All other effects and interactions were not significant. After MCAo, no significant interaction was found for PaO2 (P>0.24) but the factors precluded some significant effects (anaesthetic: P=0.0008; duration: P<0.0001). A significant interaction is obtained (P<0.0008) for pH. Therefore, for each factor level, a Students t-test was performed. All comparisons were significant for pH (P<0.04) and the largest difference between the groups was 0.07 pH units.
|
Continuous MAP recordings were averaged over 20 min periods (Fig. 4). MAP was analysed by a three-way repeated measures ANOVA (anaesthetic, duration of anaesthesia, time). A second order interaction was found (P<0.0001) and hence a two-way factorial ANOVA was performed for each time. Before ischaemia, only the anaesthetic factor was significant effect (P<0.022). For the first time period after MCAo, a significant interaction was obtained (P<0.0015). For both anaesthetics, a significant effect was obtained for the duration factor (P<0.012). There was also a significant difference between short-duration halothane and short-duration desflurane groups (P=0.001). For all other times after MCAo, only the duration factor was significant (P<0.0001).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although clinical evidence is lacking, volatile anaesthetics have been shown to provide neuroprotective effects against ischaemia in various species and experimental models.15 It has been shown that 1.4 MAC of halothane or sevoflurane, as compared with the awake state, reduces infarct volume by approximately 72 and 57%, respectively, in rats exposed to 90 min of focal cerebral ischaemia.2 In the same model, 1.0 MAC of halothane remains neuroprotective even when brain temperature is maintained in normothermic conditions.3 Our results are in accordance with these findings in that the long-duration administration of halothane 1.11.2% during ischaemia in the present study provided a significant decrease in cerebral infarct volume in normothermic rats. Several experiments with isoflurane have shown that this compound (0.5, 1.0 MAC) protects the brain in rat models of global ischaemia4 9 but the protection against focal cerebral ischaemia is debatable. In comparison with nitrous oxide/fentanyl, isoflurane 2.5% reduces infarct volume and the frequency of transient ischaemic depolarizations in a 2 h MCAo rat model.1 It has been reported that 1.5 MAC isoflurane delays but does not prevent cerebral infarction in rats subjected to a 70 min focal ischaemia.10 Finally, in spontaneously hypertensive rats, infarct volume following 3 h of MCAo and 2 h of reperfusion in animals anaesthetized with isoflurane were larger than those in animals anaesthetized with halothane.11 Although desflurane improves neurological outcome following transient global cerebral ischaemia in rats4 and increases tissue oxygenation during transient MCAo in humans,5 no studies have been reported on the effects of desflurane on histopathology following focal ischaemia.
Physiological parameters are unlikely to be responsible for the differences in infarct size observed between long-duration and short-duration groups. Indeed, there was a significantly higher MAP in the short-duration groups as compared with the long-duration groups. As acute increases in arterial pressure decrease brain injury,12 one would expect smaller infarcts in the short-duration anaesthetized groups. The differences between groups concerning the other physiological parameters (i.e. PaO2, PaCO2, pH and rectal and pericranial temperatures) were minor and probably without any biological significance. The decrease in infarct volume in the long-duration groups may, therefore, be reasonably attributed to the inhalation of nitrous oxide and/or the volatile anaesthetics.
Nitrous oxide has been reported to have no effect or to increase cerebral ischaemic damage. In a 2-h rat reversible MCAo model, nitrous oxide 70% does not modify infarct volume.13 In a rat brain global ischaemia model, animals ventilated with nitrous oxide 70% and oxygen 30% (as compared with those ventilated with nitrogen 70% and oxygen 30%) had a worse outcome.9 Other authors have shown that nitrous oxide 50% impairs electrophysiologic recovery after 3.5 min of hypoxia in rat hippocampal slices.14 Therefore, in the present study, nitrous oxide is probably not responsible for the observed neuroprotection.
Volatile anaesthetics may decrease ischaemic damage by various mechanisms. First, volatile anaesthetics produce cerebral vasodilation. Halothane potently dilates intracerebral arterioles in the rat brain.15 In dogs, both isoflurane and sevoflurane significantly dilate pial arterioles, an effect mediated, at least in part, via activation of K+ATP channels.16 In patients undergoing routine spinal surgery, sevoflurane and isoflurane produce a dose-dependent cerebral vasodilatory effect.17 Considering that vasodilation might produce an increase in intra-ischaemic cerebral blood flow and an increased supply of oxygen and glucose, vasodilation by volatile anaesthetics could be neuroprotective. Secondly, 0.75 MAC halothane or isoflurane reduces cerebral metabolic rate, an effect which accompanies an improved outcome from severe brain ischaemia in the rat.18 Thirdly, volatile anaesthetics produce hypothermia which is in itself neuroprotective.19 20 Such a mechanism does not apply to our present study as body temperature was controlled. However, we cannot rule out an effect on brain temperature.
Volatile anaesthetics have major effects on synaptic neurotransmission which may participate in their neuroprotective effects, given the well described deleterious effects of glutamate during ischaemia.21 22 There is increasing evidence that general anaesthetics enhance GABA-mediated synaptic inhibition23 and that they depress excitatory glutamate-mediated transmission.24 In rat hippocampal brain slices, electrophysiological studies have shown that volatile anaesthetics depress glutamate transmission.25 Volatile anaesthetics increase glutamate uptake as demonstrated in cultured astrocytes from the hippocampi of rat embryos exposed to enflurane, isoflurane, and sevoflurane.26 Furthermore, isoflurane reduces both L-glutamate and NMDA-mediated calcium influx in rat cortical brain slices.27 Volatile anaesthetics modulate the activity of GABAA receptors by a direct action on the channel complex.23 28 29 The block of voltage-dependent Na+ channels by low concentrations of anaesthetics might have a significant role for neuroprotection because this attenuates membrane depolarization initiating the removal of the magnesium block of the NMDA receptor.30 Finally, halothane, enflurane, and isoflurane induce a dose- dependent uncoupling of gap junctions in primary cultures of mouse striatal astrocytes.31 This effect together with the effects on glutamate transmission, may partly explain why halothane inhibits spreading depression like-depolarizations following ischaemia, thus leading to a reduced infarct volume.32
Our study shows that desflurane reduces infarct volume following transient MCAo. Furthermore, this effect is greater than that induced by halothane. In our study, the concentration of halothane and desflurane during the preparation and focal cerebral ischaemia were equipotent. Therefore, the higher neuroprotection of desflurane cannot be attributed to the difference in the applied anaesthetic concentrations for the two agents.
In other respects, changes in pericranial temperature by volatile anaesthetics may influence infarct volume. When brain temperature is controlled, isoflurane 0.7% does not protect the rat brain from 75 min intraluminal MCAo.21 In contrast, the neuroprotection induced by halothane is maintained when pericranial temperature is controlled.3 Pericranial temperature correlates well with brain temperature.33 In our study, no significant difference in pericranial temperature under desflurane or halothane was found and the average difference was approximately 0.12°C. Therefore, in our study, the non-significant changes in brain temperature are probably not responsible for the higher neuroprotection conferred by desflurane.
The increase in brain tissue partial oxygen pressure and decrease in brain acidosis induced by desflurane may participate in its neuroprotective effect during MCAo.5 Although desflurane possesses most of the properties of the other volatile anaesthetics discussed above, it also induces a concentration-dependent biphasic effect on sympathetic activity. Indeed, with concentrations of desflurane under 6%, sympathetic activity is increased, while desflurane decreases sympathetic activity at higher concentrations.34 Thus, given the concentrations used in the present study, it may be assumed that desflurane reduces sympathetic activity and catecholamine release. In this respect, Engelhard and colleagues4 demonstrated that the neurological improvement induced by desflurane following global ischaemia was associated with a reduction in sympathetic activity and catecholamine release. Several studies have addressed the effects of catecholamines in cerebral ischaemia. In a rat model of incomplete ischaemia, ganglionic block decreases plasma catecholamine concentrations and improves neurologic outcome.35 In addition, neurologic outcome and stroke-related mortality were worse in rats with increased plasma epinephrine and norepinephrine concentrations compared with rats with ganglionic block.36 Moreover, pre-ischaemic depletion of brain norepinephrine decreases infarct size and improves neurologic outcome in normothermic rats.37 It is clear that sympathetic activity and adrenergic receptors modulate brain damage but the brain protection mechanisms suggested here against cerebral ischaemia remain largely unknown.
In conclusion, in our experimental methods, we have shown that desflurane affords a more effective protection than halothane against transient focal cerebral ischaemia in rats. Although clinical evidence is lacking, numerous experimental studies support the hypothesis that volatile anaesthetics are neuroprotective following cerebral ischaemia.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 Warner DS, McFarlane C, Todd MM, Ludwig P, McAllister AM. Sevoflurane and halothane reduce focal ischaemic brain damage in the rat. Possible influence on thermoregulation. Anesthesiology 1993; 79: 98592[ISI][Medline]
3 Warner DS, Ludwig PS, Pearlstein R, Brinkhous AD. Halothane reduces focal ischaemic injury in the rat when brain temperature is controlled. Anesthesiology 1995; 82: 123745[CrossRef][ISI][Medline]
4 Engelhard K, Werner C, Reeker W, et al. Desflurane and isoflurane improve neurological outcome after incomplete cerebral ischaemia in rats. Br J Anaesth 1999; 83: 41521
5 Hoffman WE, Charbel FT, Edelman G, Ausman JI. Thiopental and desflurane treatment for brain protection. Neurosurgery 1998; 43: 10503[ISI][Medline]
6 Orliaguet G, Vivien B, Langeron O, Bouhemad B, Coriat P, Riou B. Minimum alveolar concentration of volatile anaesthetics in rats during postnatal maturation. Anesthesiology 2001; 95: 7349[CrossRef][ISI][Medline]
7 Eger EI, Johnson BH. MAC of I-653 in rats, including a test of the effect of body temperature and anaesthetic duration. Anesth Analg 1987; 66: 9746[Abstract]
8 Zea longa E, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 1989; 20: 8491[Abstract]
9 Baughman VL, Hoffman WE, Thomas C, Albrecht RF, Miletich DJ. The interaction of nitrous oxide and isoflurane with incomplete cerebral ischaemia in the rat. Anesthesiology 1989; 70: 76774[ISI][Medline]
10 Kawaguchi M, Kimbro JR, Drummond JC, Cole DJ, Kelly PJ, Patel PM. Isoflurane delays but does not prevent cerebral infarction in rats subjected to focal ischaemia. Anesthesiology 2000; 92: 133542[ISI][Medline]
11 Drummond JC, Cole DJ, Patel PM, Reynolds LW. Focal cerebral ischaemia during anaesthesia with etomidate, isoflurane, or thiopental: a comparison of the extent of cerebral injury. Neurosurgery 1995; 37: 7428[ISI][Medline]
12 Hayashi S, Nehls DG, Kieck CF, Vielma J, Degirolami U, Crowell RM. Beneficial effects of induced hypertension on experimental stroke in awake monkeys. J Neurosurg 1984; 60: 1517[ISI][Medline]
13 Warner DS, Zhou JG, Ramani R, Todd MM, McAllister AM. Nitrous oxide does not alter infarct volume in rats undergoing reversible middle cerebral artery occlusion. Anesthesiology 1990; 73: 68693[ISI][Medline]
14 Amorim P, Chambers G, Cottrell J, Kass IS. Nitrous oxide impairs electrophysiologic recovery after severe hypoxia in rat hippocampal slices. Anesthesiology 1997; 87: 64251[ISI][Medline]
15 Staunton M, Drexler C, Schmid PG, Havlik HS, Hudetz AG, Farber NE. Neuronal nitric oxide synthase mediates halothane-induced cerebral microvascular dilation. Anesthesiology 2000; 92: 12532[ISI][Medline]
16 Iida H, Ohata H, Iida M, Watanabe Y, Dohis S. Isoflurane and sevoflurane induce vasodilation of cerebral vessels via ATP-sensitive K+ channel activation. Anesthesiology 1998; 89: 95460[ISI][Medline]
17 Matta BF, Heath KJ, Tipping K, Summors AC. Direct cerebral vasodilatory effects of sevoflurane and isoflurane. Anesthesiology 1999; 91: 67780[CrossRef][ISI][Medline]
18 Nellgard B, Mackensen GB, Pineda J, Wellons JC, Pearlstein RD, Warner DS. Anaesthetic effects on cerebral metabolic rate predict histologic outcome from near-complete forebrain ischaemia in the rat. Anesthesiology 2000; 93: 4316[CrossRef][ISI][Medline]
19 Corbett D, Thornhill J. Temperature modulation (hypothermic and hyperthermic conditions) and its influence on histological and behavioral outcomes following cerebral ischaemia. Brain Pathol 2000; 10: 14552[ISI][Medline]
20 Hoffman WE, Thomas C. Effects of graded hypothermia on outcome from brain ischaemia. Neurol Res 1996; 18: 1859[ISI][Medline]
21 Sarraf-Yazdi S, Sheng H, Miura Y, et al. Relative neuroprotective effects of dizocilpine and isoflurane during focal cerebral ischaemia in the rat. Anesth Analg 1998; 87: 728[Abstract]
22 Buchan AM, Slivka A, Xue D. The effect of the NMDA receptor antagonist MK-801 on cerebral blood flow and infarct volume in experimental focal stroke. Brain Res 1992; 574: 1717[CrossRef][ISI][Medline]
23 Tanelian DL, Kosek P, Mody I, MacIver MB. The role of the GABAA receptor/chloride channel complex in anaesthesia. Anesthesiology 1993; 78: 75776[ISI][Medline]
24 Perouansky M, Baranov D, Salman M, Yaari Y. Effects of halothane on glutamate receptor-mediated excitatory postsynaptic currents. A patch-clamp study in adult mouse hippocampal slices. Anesthesiology 1995; 83: 10919[ISI][Medline]
25 Maclver MB, Mikulec AA, Amagasu SM, Monroe FA. Volatile anaesthetics depress glutamate transmission via presynaptic actions. Anesthesiology 1996; 85: 82334[CrossRef][ISI][Medline]
26 Miyazaki H, Nakamura Y, Arai T, Kataoka K. Increase of glutamate uptake in astrocytes: a possible mechanism of action of volatile anaesthetics. Anesthesiology 1997; 86: 135966[ISI][Medline]
27 Bickler PE, Buck LT, Hansen BM. Effects of isoflurane and hypothermia on glutamate receptor-mediated calcium influx in brain slices. Anesthesiology 1994; 81: 14619[ISI][Medline]
28 Li X, Czajkowski C, Pearce RA. Rapid and direct modulation of GABAA receptors by halothane. Anesthesiology 2000; 92: 136675[ISI][Medline]
29 Jenkins A, Greenblatt EP, Faulkner HJ, et al. Evidence for a common binding cavity for three general anaesthetics within the GABAA receptor. J Neurosci 2001; 21: RC136[Medline]
30 Ratnakumari L, Hemmings HCJ. Inhibition of presynaptic sodium channels by halothane. Anesthesiology 1998; 88: 104354[ISI][Medline]
31 Mantz J, Cordier J, Giaume C. Effects of general anaesthetics on intercellular communications mediated by gap junctions between astrocytes in primary culture. Anesthesiology 1993; 78: 892901[ISI][Medline]
32 Saito R, Graf R, Hubel K, Fujita T, Rosner G, Heiss WD. Reduction of infarct volume by halothane: effect on cerebral blood flow or perifocal spreading depression-like depolarizations. J Cereb Blood Flow Metab 1997; 17: 85764[CrossRef][ISI][Medline]
33 Jiang JY, Lyeth BG, Clifton GL, Jenkins LW, Hamm RT, Hayes RC. Relationship between body and brain temperature in traumatically brain-injured rodents. J Neurosurg 1991; 74: 4926[ISI][Medline]
34 Pac-Soo CK, Wang C, Ma D, Chakrabarti MK, Whitwam JG. Vagally mediated sympathoexcitation and central depression by desflurane in rabbits. Br J Anaesth 2000; 84: 77782[Abstract]
35 Werner C, Hoffman WE, Thomas C, Miletich DJ, Albrecht RF. Ganglionic blockade improves neurologic outcome from incomplete ischaemia in rats: partial reversal by exogenous catecholamines. Anesthesiology 1990; 73: 9239[ISI][Medline]
36 Shu CC, Hoffman WE, Thomas C, Albrecht RF. Sympathetic activity enhances glucose-related ischaemic injury in the rat. Anesthesiology 1993; 78: 11205[ISI][Medline]
37 Nellgard B, Mackensen GB, Sarraf-Yazdi S, Miura Y, Pearlstein R, Warner DS. Pre-ischaemic depletion of brain norepinephrine decreases infarct size in normothermic rats exposed to transient focal cerebral ischaemia. Neurosci Lett 1999; 275: 16770[CrossRef][ISI][Medline]