1Neurotransmission Laboratory, Academic Department of Anaesthesia and Intensive Care, The Royal London and St Bartholomews School of Medicine and Dentistry, Royal London Hospital, Whitechapel, London E1 1BB, UK. 2Division of Biomedical Sciences, Queen Mary & Westfield College, Mile End Road, London E1 4NS, UK*Corresponding author
Accepted for publication: October 24, 2000
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Abstract |
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Br J Anaesth 2001; 86: 5504
Keywords: brain, ischaemia; anaesthetics volatile, sevoflurane
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Introduction |
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There is some evidence from animal studies that sevoflurane is neuroprotective in models of cerebral ischaemia.5 Since global ischaemic insults, and even frank infarcts, can occur in a theatre setting, a neuroprotective anaesthetic is of considerable clinical interest.
During cerebral ischaemia, a cascade of events occur that result in neuronal death. One of the earliest of these is the release of neurotransmitters, in particular the excitatory amino acids.6 Reduction of transmitter release thus represents a possible mechanism by which drugs may exert a neuroprotective effect.
Previous work in this laboratory has examined transmitter efflux in an in vitro model of cerebral ischaemia based on brain slices.7 We have found that various recognized neuroprotective agents reduce neurotransmitter efflux. In particular, we recently found that halothane slowed ischaemia-induced dopamine efflux.8 In the present study we examined the effects of sevoflurane on the efflux of dopamine, glutamate and aspartate.
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Materials and methods |
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Standard aCSF (pH 7.4) was used, consisting of (mmol litre1) NaCl (126.0), KCl (4.0), KH2PO4 (1.4), MgSO4 (1.3), CaCl2 (2.4), NaHCO3 (26.0), (+)-glucose (4.0) and ascorbic acid (0.4). The glucose concentration was reduced to 2 mmol litre1 during the induction of ischaemia.
Induction of ischaemia
After recovery in the saver, the slices were transferred to a submersion-type recording chamber, modified as previously described7, and superfused with aCSF for 30 min before induction of ischaemia. For the measurement of dopamine, single slices were placed in the chamber and perfused with aCSF at 400 ml h1. For excitatory amino acid experiments, four slices were carefully chosen so as to allow comparison between sets of slices. In these experiments, slices were used within 90 min of cutting and four slices were then superfused together in a single chamber at 300 ml h1, in order to enhance the detection of glutamate and aspartate efflux.
The superfusion system comprised separate flasks of maintenance or ischaemic aCSF, gassed with 95% oxygen/5% carbon dioxide or 95% nitrogen/5% carbon dioxide, respectively, and pumped into the slice chamber by a Watson-Marlow 302M peristaltic pump via gas-impermeable (Marprene) tubing. The temperature of the slice chamber was maintained at 34.0 (0.2)°C throughout. After 30 min of superfusion with maintenance aCSF, superfusion with ischaemic aCSF was initiated for 30 min.
Sevoflurane was applied to the brain slices at 4% (approximately 1.7 MAC10) throughout the experiment (during both pre-incubation in maintenance aCSF and imposition of ischaemia) via a calibrated Blease Datum vaporizer.
Detection of dopamine efflux
Ischaemia-induced dopamine efflux was measured using fast cyclic voltammetry.7 Carbon fibre microelectrodes, 7 µm in diameter, were positioned in the dorsolateral zone of the striatum, under micromanipulator control. Auxiliary (stainless steel) and reference (Ag/AgCl) electrodes were located at convenient points in the slice chamber where they would not obstruct the voltammetric or d.c. potential electrodes. Voltammetric scans (1.0 to +1.4 V compared with reference electrodes, 480 V s1) were performed every 4 s with a Millar Voltammetric Analyser (PD Systems, West Molesey, UK). A sample-and-hold record of current at the peak dopamine oxidation voltage (+0.6 V compared with reference electrodes) was displayed on a chart recorder while entire voltammetric scans were recorded on a digital storage oscilloscope (Nicolet 310 Series) and saved on to floppy disk. At the end of the experiment, the carbon fibre microelectrodes were calibrated by standard flow injection analysis (400 ml h1) in solutions of dopamine (500 µl of 100 µmol litre1).
Three dopamine efflux variables were measured: the time from initiation of ischaemia to the onset of dopamine efflux (ton, in s), the maximum extracellular dopamine concentration (DAmax, in µmol litre1) and the mean rate of dopamine efflux from ton to DAmax (DA/
t, in µmol litre1 s1).
Detection of excitatory amino acid efflux
At 5 min intervals throughout the experiment, a 1 min sample (5 ml) of slice superfusate was collected into glass vials and frozen until analysed for glutamate and aspartate content. Glutamate and aspartate were determined using a modification of the HPLC method of Smolders and colleagues.11 The method is based on the derivatization of amino acids using o-phthaldialdehyde (OPA). A stock solution of 120 mg OPA in 6 ml methanol was prepared. Derivatizing reagent was prepared by mixing 0.6 ml OPA solution with 5.4 ml borate buffer (pH 9.0) and 60 µl 2-mercaptoethanol. Equal volumes (50 µl) of superfusates collected from slices and derivatizing reagent were mixed at room temperature (2426°C) and, exactly 2 min later, 50 µl were injected into the chromatograph.
The OPA derivatives were separated on a 100x3.2 mm i.d. HIRPB column (HiChrom, Reading, UK) eluted with 10% HPLC-grade acetonitrile in 0.1 M sodium acetate buffer (pH 6.0) at 1 ml min1 with an Altex 110 pump (Beckman, High Wycombe, UK). The compounds were detected on a PerkinElmer LS-4 fluorimeter (Beaconsfield, UK) with the excitation monochromator set at 350 nm and the emission set at 450 nm. Peaks were recorded on an analogue recorder or digitally using a 16 bit A/D converter (Jones Chromatography, Hengoed, UK) in an ICL M55 computer. Four minutes after the injection, the column was washed with 90% acetonitrile in water for 1 min, using a second Altex pump and an electronic valve actuator (Jones Chromatography) controlled from the computer. The column was equilibrated with eluent for 5 min before the next injection. Calibration curves were generated using standard solutions in water containing 1251000 nmol litre1 glutamate and aspartate and data were expressed as nmol of amino acid litre1 superfusate per slice.
For estimation of variability, calibration solutions were prepared in the range 10.031 µmol litre1, plus blank. Estimates of the intra- and inter-assay precision and accuracy values were obtained by quintuplicate analyses of solutions containing 1, 0.5, 0.2, 0.1, 0.05 and 0.02 µmol litre1 of aspartate and glutamate. Intra-assay coefficients of variation of replicate analyses of glutamate and aspartate concentrations were between 2 and 6%. The lower limit of quantification was taken as 0.05 µmol litre1 as coefficients of variation for samples at 0.02 µmol litre1 were >20%.
Drugs and chemicals
All chemicals used to make the aCSF were of standard AnalaR grade from BDH Lab supplies (Poole, UK). Dopamine was obtained from Sigma (Poole, UK). Sevoflurane was a gift from Abbott Laboratories Ltd.
Data analysis
The effects of sevoflurane on ischaemia-induced excitatory amino acid efflux were calculated as percentages of pre-ischaemia values and were compared with control by two-way analysis of variance (ANOVA). The effects of sevoflurane on basal (pre-ischaemic) glutamate and aspartate efflux were analysed by unpaired t-tests. The effects of sevoflurane on individual measures of dopamine efflux were tested using paired t-tests.
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Results |
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Basal excitatory amino acid efflux in the superfusate was not significantly different in control slices and those treated with 4% sevoflurane (Table 1). The effects of ischaemia on excitatory amino acid efflux were very different from those on dopamine efflux. Figure 2 shows the effect of sevoflurane on ischaemia-induced glutamate efflux. After imposition of ischaemia, there was a significant (P<0.001) time-dependent increase in glutamate efflux over the 30 min course of the ischaemic episode. This was significantly reduced in the presence of 4% sevoflurane (P<0.001; two-way ANOVA).
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Discussion |
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An alternative approach to neuroprotection, rather than blocking postsynaptic actions, is to prevent the efflux of neurotransmitter. Studies by our group have shown that ischaemia-induced dopamine efflux may be attenuated or delayed by several agents or strategies with neuroprotective potential. These include blockers of calcium and sodium channels,14,15 NMDA antagonists16 and hypothermia.17
Efflux of glutamate and aspartate in ischaemia is sometimes held to be mediated by reversal of the glutamate carrier.18 The mechanism of dopamine efflux is less clear. Whilst at least one group has suggested that it too occurs through reversal of the transporter,19 we found no effect of dopamine uptake inhibitors on ischaemia-induced dopamine efflux and thus suggested that its primary mode of release was exocytotic.7
The possibility that general anaesthetics are neuroprotective is widely promoted.20 Although ketamine is known to be neuroprotective, presumably because of its capacity to block NMDA receptors, it is possible that others with less overt actions may also be protective. We have recently shown that halothane is able to slow dopamine efflux in this model.8
In the present study, we found that sevoflurane not only slowed ischaemia-induced dopamine efflux in a manner similar to halothane but also reduced release of glutamate and aspartate. The effect on aspartate was particularly striking. However, since aspartate binds to NMDA receptors with approximately a tenth of the affinity of glutamate,21 such a large reduction in its release is likely to have less neuroprotective consequences than the diminution of glutamate release.
It should be said that in measuring dopamine efflux by voltammetry, we have examined the effect of sevoflurane on ischaemia-induced transmitter release from striatal terminals, whereas with the method for investigating excitatory amino acid efflux, we have examined transmitter release from cells and synaptic terminals in both the cortex and striatum. It would be interesting to discover if the differential effect of sevoflurane on maximal ischaemia-induced transmitter release lies in this different neuroanatomical source of transmitter or whether it reflects true differences in the mode of action of the anaesthetic on the different transmitter systems per se. Such questions might be resolved by electrode-based techniques, but these have not yet reached the technological level required for such studies.
Sevoflurane has been reported to be an effective neuroprotective agent in cerebral ischaemia. For instance, Werner and colleagues22 found that sevoflurane improved outcome after a focal ischaemic insult in rats. Sevoflurane also expedites the recovery of brain energy metabolism, relative to halothane, in global ischaemia.23
In common with other volatile anaesthetics, sevoflurane has many biochemical actions. It blocks nicotinic receptors24 whilst enhancing the effects of agonist stimulation at -amino-butyrate (GABA)A and GABAB receptors.25 Recently, Li and Pearce26 showed that halothane enhanced the effects of GABA at GABAA receptors by slowing the dissociation of the agonist; they suggested that this may be a major mode of action of other volatile anaesthetic agents. Also of interest in the context of the present report, a recent study found that sevoflurane enhanced glutamate uptake by astrocytes.27 Such an effect could contribute to the decreased efflux not only of glutamate observed here but also of aspartate, which is also a substrate for the transporter.28
The volatile anaesthetics have also been shown to activate potassium channels whilst blocking sodium channels in rat brain slice preparations.29 We have previously shown that sodium channel blockade slows ischaemia-induced dopamine efflux in striatal slices,15 so it is possible that such a mechanism underlies some of the effects of sevoflurane observed here.
Throughout this study, sevoflurane administration was initiated before the ischaemic insult. Although this has little bearing on the treatment of stroke, where neuroprotection can only be administered in a post-ictal manner, such a circumstance would be analogous to that found in the operating theatre, where an ischaemic episode might occur under anaesthesia. Surgery for carotid endarterectomy or coronary artery bypass grafting carries a significant risk of cerebral ischaemia, so there is a role for prior administration of appropriate neuroprotectants. The results of the present study show that sevoflurane, at a clinically relevant concentration (approximately 1.7 MAC10), can reduce the efflux of neurotoxic transmitters under such conditions and thus may be neuroprotective.
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Acknowledgement |
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