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Effects of sevoflurane on dopamine, glutamate and aspartate release in an in vitro model of cerebral ischaemia

2001; Elsevier BV; Volume: 86; Issue: 4 Linguagem: Inglês

10.1093/bja/86.4.550

1471-6771

C. C. Toner, Kim A. Connelly, Robin Whelpton, Satinder Bains, Adina T. Michael‐Titus, Daniel P. McLaughlin, Jon Stamford,

Anesthesia and Neurotoxicity Research

Release of excitatory amino acids and dopamine plays a central role in neuronal damage after cerebral ischaemia. In the present study, we used an in vitro model of ischaemia to investigate the effects of sevoflurane on dopamine, glutamate and aspartate efflux from rat corticostriatal slices. Slices were superfused with artificial cerebrospinal fluid at 34°C and episodes of 'ischaemia' were mimicked by removal of oxygen and reduction in glucose concentration from 4 to 2 mmol litre−1 for ≤30 min. Dopamine efflux was monitored in situ by voltammetry while glutamate and aspartate concentrations in samples of the superfusate were measured by HPLC with fluorescence detection. Neurotransmitter outflow from slices was measured in the absence or presence of sevoflurane (4%). After induction of ischaemia in control slices, there was a mean (sem) delay of 166 (7) s (n=5) before sudden efflux of dopamine which reached a maximum extracellular concentration of 77.0 (15.2) µmol litre−1. Sevoflurane (4%) reduced the rate of dopamine efflux during ischaemia (6.90 (1.5) and 4.73 (1.76) µmol litre−1 s−1 in controls and sevoflurane-treated slices, respectively; P<0.05), without affecting its onset or magnitude. Excitatory amino acid efflux was much slower. Ischaemia-induced glutamate efflux had not reached maximum after 30 min of ischaemia. Basal (pre-ischaemic) glutamate and aspartate efflux per slice was 94.8 (24.8) and 69.3 (31.5) nmol litre−1 superfusate (n=4) and was not significantly reduced by 4% sevoflurane. Ischaemia greatly increased glutamate and aspartate efflux (to a maximum of 919 (244)% and 974 (489)% of control, respectively). However, ischaemia-induced efflux of both glutamate and aspartate was significantly reduced by 4% sevoflurane (P<0.001 for glutamate, P<0.01 for aspartate). In summary, sevoflurane may owe part of its reported neuroprotective effect to a reduction of ischaemia-induced efflux of excitatory amino acids and, to a lesser extent, dopamine. Release of excitatory amino acids and dopamine plays a central role in neuronal damage after cerebral ischaemia. In the present study, we used an in vitro model of ischaemia to investigate the effects of sevoflurane on dopamine, glutamate and aspartate efflux from rat corticostriatal slices. Slices were superfused with artificial cerebrospinal fluid at 34°C and episodes of 'ischaemia' were mimicked by removal of oxygen and reduction in glucose concentration from 4 to 2 mmol litre−1 for ≤30 min. Dopamine efflux was monitored in situ by voltammetry while glutamate and aspartate concentrations in samples of the superfusate were measured by HPLC with fluorescence detection. Neurotransmitter outflow from slices was measured in the absence or presence of sevoflurane (4%). After induction of ischaemia in control slices, there was a mean (sem) delay of 166 (7) s (n=5) before sudden efflux of dopamine which reached a maximum extracellular concentration of 77.0 (15.2) µmol litre−1. Sevoflurane (4%) reduced the rate of dopamine efflux during ischaemia (6.90 (1.5) and 4.73 (1.76) µmol litre−1 s−1 in controls and sevoflurane-treated slices, respectively; P<0.05), without affecting its onset or magnitude. Excitatory amino acid efflux was much slower. Ischaemia-induced glutamate efflux had not reached maximum after 30 min of ischaemia. Basal (pre-ischaemic) glutamate and aspartate efflux per slice was 94.8 (24.8) and 69.3 (31.5) nmol litre−1 superfusate (n=4) and was not significantly reduced by 4% sevoflurane. Ischaemia greatly increased glutamate and aspartate efflux (to a maximum of 919 (244)% and 974 (489)% of control, respectively). However, ischaemia-induced efflux of both glutamate and aspartate was significantly reduced by 4% sevoflurane (P<0.001 for glutamate, P 20%. 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. 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. Induction of a period of ischaemia (hypoxia/hypoglycaemia) in slices at 34°C resulted in a consistent pattern of striatal dopamine efflux in untreated control tissue. After an initial period of quiescence lasting 2–3 min, there was a sudden efflux of dopamine, typically reaching a peak extracellular concentration of 50–100 µmol litre−1 within 20 s before falling slowly back towards baseline over a period of several minutes. Figure 1 shows a representative profile of dopamine efflux in control and sevoflurane-treated striatal slices. Although the onset of dopamine efflux appeared longer in 4% sevoflurane (mean (sem) ton 166 (7) s in controls, 211 (64) s in sevoflurane-treated slices; n=5), this was not significant (note the truncated x-axis in Figure 1). Sevoflurane did not significantly reduce the maximum level of dopamine efflux (DAmax 77.0 (15.2) and 67.8 (12.4) µmol litre−1 in controls and sevoflurane-treated slices, respectively). However, the rate of dopamine efflux was significantly lower in slices exposed to sevoflurane (δDA/δt 6.90 (1.5) and 4.73 (1.76) µmol litre−1 s−1 in controls and sevoflurane-treated slices, respectively; P<0.01). 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).Table 1Basal excitatory amino acid efflux from corticostriatal slices. Mean (SEM), n=4 (controls), n=5 (sevoflurane)Amino acidBasal amino acid efflux per slice (nmol litre–1superfusate)Controls4% SevofluraneAspartate69.3 (31.5)42.5 (12.8)Glutamate94.8 (24.8)66.5 (18.3) Open table in a new tab A similar profile was observed with aspartate. The effect of sevoflurane on ischaemia-induced aspartate efflux is shown in Figure 3. As with glutamate, there was a substantial increase in aspartate efflux over the duration of the ischaemic episode. This was essentially abolished in the presence of 4% sevoflurane (P<0.01; two-way ANOVA). The role of excitatory amino acids in ischaemic damage is well established. Efflux of glutamate and/or aspartate is an early event in a cascade that leads to neuronal death and drugs that block the postsynaptic actions of excitatory amino acids at N-methyl-d-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors have been shown to be neuroprotective in ischaemia.12McCulloch J Glutamate receptor antagonists in cerebral ischaemia.J Neural Transm. 1994; 43: 71-79Google Scholar However, glutamate is only one of many transmitters released in ischaemia. Recent reports have suggested that dopamine released in ischaemia may also mediate neuronal damage, at least in the striatum and at least partly through an interaction with D2 receptors.13Hashimoto N Matsumoto T Mabe H Hashitani T Nishino H Dopamine has inhibitory and accelerating effects on ischaemia-induced neuronal damage in the rat striatum.Brain Res Bull. 1994; 33: 281-288Crossref PubMed Scopus (49) Google Scholar 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,14Toner CC Stamford JA Involvement of N- and P/Q- but not L- or T-type voltage-gated calcium channels in ischaemia-induced striatal dopamine release in vitro.Brain Res. 1997; 748: 85-92Crossref PubMed Scopus (32) Google Scholar,15Toner CC Stamford JA Sodium channel blockade unmasks two temporally distinct mechanisms of striatal dopamine release during hypoxia/hypoglycaemia in vitro.Neuroscience. 1997; 81: 999-1007Crossref PubMed Scopus (22) Google Scholar NMDA antagonists16Toner CC Stamford JA Characteristics of the NMDA receptor modulating hypoxia/hypoglycaemia-induced rat striatal dopamine release in vitro.Eur J Pharmacol. 1997; 340: 133-143Crossref PubMed Scopus (15) Google Scholar and hypothermia.17Toner CC Stamford JA Effects of metabolic alterations on dopamine release in an in vitro model of neostriatal ischaemia.Brain Res Bull. 1997; 48: 395-399Crossref Scopus (20) Google Scholar Efflux of glutamate and aspartate in ischaemia is sometimes held to be mediated by reversal of the glutamate carrier.18Takahashi M Billups B Rossi D Sarantis M Hamann M Attwell D The role of glutamate transporters in glutamate homeostasis in the brain.J Exp Biol. 1997; 200: 401-409Crossref PubMed Google Scholar The mechanism of dopamine efflux is less clear. Whilst at least one group has suggested that it too occurs through reversal of the transporter,19Kim KW Kim DC Kim YH Eun YA Kim HI Cho KP Ca2+-dependent and independent mechanisms of ischaemia-evoked release of [3H]-dopamine from rat striatal slices.Clin Exp Pharmacol Physiol. 1995; 22: 301-302Crossref PubMed Scopus (21) Google Scholar we found no effect of dopamine uptake inhibitors on ischaemia-induced dopamine efflux and thus suggested that its primary mode of release was exocytotic.7Toner CC Stamford JA Real time measurement of dopamine release in an in vitro model of neostriatal ischaemia.J Neurosci Methods. 1996; 67: 133-140PubMed Google Scholar The possibility that general anaesthetics are neuroprotective is widely promoted.20Toner CC Stamford JA General anaesthetics as neuroprotective agents.in: Stamford JA Strunin L Neuroprotection. Baillière Tindall, London1996: 515-533Abstract Full Text PDF Scopus (9) Google Scholar 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.8Toner CC Stamford JA Effects of halothane on ischaemia-induced striatal dopamine release in vitro.Eur J Neurosci. 1998; 10: 96Google Scholar 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,21Olverman HJ Jones AW Watkins JC [3H]d-2-Amino-5-phosphonopentanoate as a ligand for N-methyl-d-aspartate receptors in the mammalian central nervous system.Neuroscience. 1988; 261: 1-15Crossref Scopus (48) Google Scholar 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 colleagues22Werner C Möllenberg O Kochs E Schulte J Sevoflurane improves neurological outcome after incomplete cerebral ischaemia in rats.Br J Anaesth. 1995; 75: 756-760Crossref PubMed Scopus (61) Google Scholar 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.23Nakajima Y Moriwaki G Ikeda K Fujise Y The effects of sevoflurane on recovery of brain energy metabolism after cerebral ischemia in the rat: a comparison with isoflurane and halothane.Anesth Analg. 1997; 85: 593-599Crossref PubMed Google Scholar In common with other volatile anaesthetics, sevoflurane has many biochemical actions. It blocks nicotinic receptors24Scheller M Bufler J Schneck H Kochs E Franke C Isoflurane and sevoflurane interact with the nicotinic acetylcholine receptor channels in micromolar concentrations.Anesthesiology. 1997; 86: 118-127Crossref PubMed Scopus (59) Google Scholar whilst enhancing the effects of agonist stimulation at γ-amino-butyrate (GABA)A and GABAB receptors.25Hirota K Roth SH Sevoflurane modulates both GABAA and GABAB receptors in area CA1 of rat hippocampus.Br J Anaesth. 1997; 78: 60-65Crossref PubMed Scopus (45) Google Scholar Recently, Li and Pearce26Li X Pearce RA Effects of halothane on GABAA receptor kinetics: evidence for slowed agonist unbinding.J Neurosci. 2000; 20: 899-907PubMed Google Scholar 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.27Miyazaki H Nakamura Y Arai T Kataoka K Increase of glutamate uptake in astrocytes'a possible mechanism of action of volatile anesthetics.Anesthesiology. 1997; 86: 1359-1366Crossref PubMed Scopus (94) Google Scholar 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.28Duan S Anderson CM Stein BA Swanson RA Glutamate induces rapid upregulation of astrocyte glutamate transport and cell-surface expression of GLAST.J Neurosci. 1999; 19: 10193-10200Crossref PubMed Google Scholar The volatile anaesthetics have also been shown to activate potassium channels whilst blocking sodium channels in rat brain slice preparations.29Sirois JE Pancrazio JJ Lynch C Bayliss DA Multiple ionic mechanisms mediate inhibition of rat motoneurones by inhalation anaesthetics.J Physiol. 1998; 512: 851-862Crossref PubMed Scopus (80) Google Scholar We have previously shown that sodium channel blockade slows ischaemia-induced dopamine efflux in striatal slices,15Toner CC Stamford JA Sodium channel blockade unmasks two temporally distinct mechanisms of striatal dopamine release during hypoxia/hypoglycaemia in vitro.Neuroscience. 1997; 81: 999-1007Crossref PubMed Scopus (22) Google Scholar 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 MAC10Crawford MW Lerman J Pilato M Orrego H Saldivia V Carmichael FJ Haemodynamic and organ blood flow responses to sevoflurane during spontaneous ventilation in the rat: a dose–response study.Can J Anaesth. 1992; 39: 270-276Crossref PubMed Scopus (37) Google Scholar), can reduce the efflux of neurotoxic transmitters under such conditions and thus may be neuroprotective. Sandip Bains held a Wellcome Vacation Scholarship.