1 Magill Department of Anaesthesia, Chelsea and Westminster Hospital and 2 Biophysics Group, The Blackett Laboratory, Imperial College of Science, Technology and Medicine, London, UK
*Corresponding author: Department of Anaesthetics and Intensive Care, Faculty of Medicine, Imperial College, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK Declaration of interest. Professor Maze and Professor Franks are Board members of an Imperial College spin-out company (Potexeon Ltd) that is interested in developing clinical applications for medical gases, including xenon. Both Professor Franks and Professor Maze are paid consultants in this activity. In addition, Air Products have funded, and continue to fund, work in the authors laboratories that bears on the actions of xenon as an anaesthetic and neuroprotectant and Air Products has a financial stake in Protexeon Ltd. However, none of the work described in this manuscript was funded by either company.
Accepted for publication: July 1, 2002
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Abstract |
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Methods. We used an in vivo rat model of brain injury in which N-methyl-DL-aspartic acid (NMA) is injected subcutaneously (s.c.) and c-Fos expression in the arcuate nucleus is used as a measure of injury. To examine the neurotoxic potential of each of the three anaesthetics with NMDA receptor antagonist properties, c-Fos expression in the posterior cingulate and retrosplenial (PC/RS) cortices was measured.
Results. Xenon dose-dependently suppressed NMA-induced c-Fos expression in the arcuate nucleus with an IC50 of 47 (2)% atm. At the highest concentration tested (75% atm) NMA-induced neuronal injury was decreased by as much as that observed with the prototypical NMDA antagonist MK801 (0.5 mg kg1 s.c.). Both nitrous oxide and ketamine dose-dependently increased c-Fos expression in PC/RS cortices; in contrast, xenon produced no significant effect. If the dopamine receptor antagonist haloperidol was given before either nitrous oxide or ketamine, their neurotoxic effects were eliminated.
Conclusions. Uniquely amongst anaesthetics with known NMDA receptor antagonist action, xenon exhibits neuroprotective properties without co-existing neurotoxicity. The reason why ketamine and nitrous oxide, but not xenon, produce neurotoxicity may involve their actions on dopaminergic pathways.
Br J Anaesth 2002; 89: 73946
Keywords: anaesthetics gases, nitrous oxide; anaesthetics gases, xenon; anaesthetics i.v., ketamine; brain, arcuade nucleus; brain, posterior cingulate and retrosplenial cortices; protein, c-Fos
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Introduction |
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Xenon, a noble gas with anaesthetic properties, has recently been found to be an NMDA receptor antagonist;8 9 another gaseous anaesthetic, nitrous oxide can also inhibit the NMDA receptor.10 Consequently, these two gases might be considered good candidates as neuroprotectants because they can be easily administered and rapidly enter the brain. However, a major deterrent to the use of NMDA antagonists as neuroprotective agents is the profound psychotomimetic behavioural changes which such drugs can produce.11 Histological data in studies conducted with the NMDA receptor antagonists ketamine, phencyclidine (PCP), and dizolcipine maleate (MK801) revealed pyramidal neuronal damage in the region of the posterior cingulate and retrosplenial (PC/RS) cortices,12 13 which may underlie these behavioural changes; similar pathological changes have been reported with nitrous oxide.14 Whether or not NMDA antagonism alone is sufficient to produce this neurotoxicity, however, is not known and it is possible that perturbation of other neurotransmitter systems is involved. For example, NMDA receptor antagonists including ketamine and nitrous oxide, can activate dopamine receptors or increase dopamine release both in vivo and in vitro.1517 Moreover, major antipsychotics with dopamine D2 receptor antagonist properties can prevent ketamines psychotomimetic side-effects.18 In addition, a recent in vitro study suggests that xenon can decrease dopamine release.19
The aims of the present study were to determine: (i) whether xenon has a neuroprotective effect in an in vivo model of neuronal excitotoxicity; (ii) whether xenon exhibits typical NMDA receptor antagonist neurotoxicity in the PC/RS cortices; and (iii) whether dopamine contributes to the neurotoxicity exhibited by anaesthetics with NMDA receptor antagonist properties. We used an immediate early gene (c-fos)-encoded protein (c-Fos), in order to assess neuronal injury as reported previously.2024
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Methods |
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Effect of xenon on NMA-induced c-Fos expression in the arcuate nucleus
Nine groups (n=34 in each group) of female SpragueDawley rats (240260 g) were treated randomly as follows: group 1 received NMA, 100 mg kg1 body wt subcutaneously (s.c.), and was exposed to 25% oxygen and 75% nitrogen; groups 25 were exposed to either 20, 40, 60, or 75% xenon plus 25% oxygen (with the remainder being nitrogen, where necessary) for 15 min before injection of NMA, 100 mg kg1 s.c.; group 6 was exposed to 25% oxygen and 75% nitrogen, and received saline, 8 ml kg1 s.c.; group 7 received 75% xenon and 25% oxygen for 15 min before injection of saline 8 ml kg1 s.c.; group 8 was exposed to 25% oxygen and 75% nitrogen; and received MK801, 0.5 mg kg1 s.c.; group 9 was exposed to 25% oxygen and 75% nitrogen; and received MK801 0.5 mg kg1 s.c., followed by NMA, 100 mg kg1 s.c. 15 min later. Doses of NMA and MK801 were selected from a previous study.14 Female animals were used because this gender has been shown to be more sensitive to excitotoxins, particularly for NMDA antagonists that are rapidly metabolized;25 for agents that are less susceptible to metabolism, such as nitrous oxide, neurotoxicity is the same for both sexes.25 Rats were randomly assigned between groups without reference to the day in their oestrous cycle.
Neurotoxicity of xenon, nitrous oxide, ketamine, and MK801
Eleven groups (n=3 in each group) of animals were treated as follows: group 1 was exposed to 25% oxygen and 75% nitrogen; groups 24 were exposed to 40, 60, or 75% xenon, plus 25% oxygen (with the remainder being nitrogen, where necessary) for 90 min; groups 57 were exposed to 40, 50, or 60% nitrous oxide, plus 25% oxygen (with the remainder being nitrogen) for 90 min; groups 810 received ketamine, 25, 50, or 100 mg kg1 s.c., respectively, and were exposed to 25% oxygen and 75% nitrogen; group 11 received MK801, 0.5 mg kg1 s.c. and was exposed to 25% oxygen and 75% nitrogen. Doses of ketamine were selected on the basis of a previous study,12 and were below those necessary to induce a loss of righting reflex.
Effect of dopamine D2 receptor antagonist haloperidol on nitrous oxide- and ketamine-induced neurotoxicity
Five groups (n=3 in each group) of animals were treated randomly as follows: group 1 received saline, 2 ml kg1 s.c. and were exposed to 75% nitrogen and 25% oxygen; group 2 received saline before exposure to 75% nitrous oxide and 25% oxygen for 90 min; group 3 received haloperidol 2.5 mg kg1 s.c., 30 min before exposure to 75% nitrous oxide and 25% oxygen for 90 min; group 4 received saline, 2 ml kg1 s.c., before ketamine 50 mg kg1 s.c.; group 5 received haloperidol 2.5 mg kg1 s.c., 30 min before ketamine 50 mg kg1 s.c.
Gas exposure
During exposure to 20, 40, 60, and 75% xenon or 40, 60, and 75% nitrous oxide as mentioned above, the gas mixture was introduced into a chamber (International Market Supply, Cheshire, UK) using calibrated flow meters. After a flush at a flow rate of 4 litres min1 for 3 min, the flow rate was reduced to 40 ml min1 for maintenance. The humidity in the chamber was maintained between 40 and 60% using silica gel (Merck, Leicestershire, UK) and the carbon dioxide level was maintained below 0.6% with soda lime.
Perfusion, brain harvesting, and tissue processing
For excitotoxicity experiments, rats were killed 3 h after NMA administration. For neurotoxicity experiments, rats were killed 90 min after exposure. Animals were deeply anaesthetized with sodium pentobarbital 100 mg kg1 i.p, perfused transcardially with 100 ml heparinized saline followed by 4% paraformaldehyde 500 ml in 0.1 M phosphate buffer. The whole brain was removed and further fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) overnight. The appropriate area of brain was sliced and embedded in paraffin. For c-Fos staining in the arcuate nucleus (excitotoxicity experiments), three sections (25 µm) were cut midway between the rostral and caudal boundaries of the arcuate nucleus. For c-Fos staining of the PC/RS cortices (neurotoxicity experiments), three sections (25 µm) were cut 6 mm caudal to the bregma at which site the maximal lesion had been noted in a previous study.14 Sections were dewaxed with 100% xylene, dehydrated with various concentrations of ethanol and finally floated in PBS for immunohistochemistry.
Immunohistochemistry
Sections were incubated for 30 min in 0.3% H2O2 in methanol in order to quench endogenous peroxidase and thereafter washed three times in PBS. Following this, the sections were incubated for 1 h in a blocking solution consisting of 3% donkey serum and 0.3% Triton X in PBS (PBT) and subsequently incubated overnight at 4°C in 1:6000 goat anti-c-Fos antibody (sc-52-G, Santa Cruz Biotechnology, Santa Cruz, CA) in PBT with 1% donkey serum. The sections were then rinsed three times with PBT and incubated with 1:200 donkey anti-goat IgG (Vector Laboratories, Burlingame, CA) in PBT with 1% donkey serum for 1 h. The sections were washed again with PBT and incubated with avidinbiotinperoxidase complex (Vector Laboratories) in PBT for 1 h. The sections were rinsed three times with PBS and stained with 3,3'-diaminobenzidine (DAB) with nickel ammonium sulphate to which hydrogen peroxide was added (DAB kit, Vector Laboratories) to achieve immunohistochemical visualization. When the staining was complete, the sections were rinsed in PBS followed by distilled water and mounted on glass slides, dehydrated with 100% ethanol, cleared with 100% xylene, and covered with cover slips. Unless otherwise mentioned, all reactions were performed at room temperature.
Quantitative counting of c-Fos neurons
The sections were photographed using a digital camera (model C2020Z, Olympus Optical, Southhall Middlesex, UK) attached to a microscope (Olympus model BX50). Under identical conditions, photographs were taken from each of the three sections per animal and counted for c-Fos positive neurons (dense black nuclear staining; see Fig. 1) by an author who was blinded to the treatment. The sum of c-Fos positive neurons for the three representative sections was the aggregate score of each animal for either the arcuate nucleus or PC/RS cortices and results are reported as mean (SEM). The statistical analysis was performed by one-way analysis of variance, followed by NewmanKeuls test. A P value <0.05 was regarded as statistically significant.
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Discussion |
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Neuroprotection by xenon
One of the aims of the present study was to use c-Fos as a marker for neuroneal injury to examine whether or not xenon could act as a neuroprotectant. We used an in vivo paradigm that involves the induction of a selective type of neuroneal injury in the arcuate nucleus of the hypothalamus following s.c. injection of a glutamate receptor agonist, NMA. Excessive activation of the NMDA subtype of the glutamate receptor produces excitotoxicity, which is implicated in both acute (e.g. stroke, seizures, hypoxia, trauma) and chronic (e.g. Huntingtons disease) neuroneal injury.2 Moreover, some NMDA receptor antagonists have shown remarkable efficacy in experimental models of neuroneal injury36 although this promise has not been translated into clinical utility. One of the possible reasons for the failure to translate this neuroprotective potential to clinical use may be the difficulty that most NMDA receptor antagonists have in penetrating the bloodbrain barrier and reaching the effect site. Xenon, however, is a small apolar atom that rapidly reaches equilibrium with the brain when present in the inspired gas. Our results show that, at concentrations that are easily attainable at normal pressures, xenon is an effective neuroprotectant (Figs 1 and 2). Indeed, at the highest concentration we tested (75% atm), which is close to the concentration required for surgery,28 29 the neuroprotection achieved was comparable with that obtained using the potent NMDA receptor antagonist MK801 (Fig. 2, inset).
Neurotoxicity of NMDA antagonists
Although NMDA receptor antagonists have neuroprotective properties, they can also cause psychotomimetic effects in humans and abnormal locomotor activities in rodents.12 30 A neuroneal correlate (although not necessarily the cause) of the ability to produce these behavioural effects is the damage which NMDA antagonists produce in PC/RS cortices12 which can be assessed by c-Fos expression.22 31 The data in Figures 3 and 4 show that, unlike nitrous oxide, ketamine and MK801, xenon had no effect on c-Fos expression in PC/RS cortices, and that concentrations of xenon that gave maximum neuroprotection (75% atm) showed no intrinsic neurotoxicity. This corroborates recent work on the neurotoxicity of nitrous oxide and ketamine14 25 and a study32 that showed nitrous oxide enhanced the neurotoxicity induced by ketamine while this was inhibited by xenon. The observation that xenon is able to counteract the neurotoxic effects of ketamine, for which there is strong evidence of NMDA receptor involvement,10 12 suggests that xenon, in addition to its ability to inhibit NMDA receptors, is likely to have additional targets.
It has been suggested that the behavioural changes produced by ketamine, phencyclidine, and MK801 in healthy individuals are similar to those seen in schizophrenia,11 which in turn has been linked to a dysfunctional dopamine system. Ketamine is known to induce cortical release of dopamine16 and D2 dopamine receptor antagonists ameliorate ketamine-induced prefrontal cortical impairment in rats.18 Like ketamine, nitrous oxide also causes dopamine metabolic changes in the cortex of rats which is seen as an increase of 3,4-dihydroxyphenylalanine, the major metabolite of dopamine in the rat brain;15 we recently confirmed this finding in a tissue culture preparation.33 In contrast, xenon does not increase dopamine release in PC12 cells.19 It is noteworthy that, of the neurotransmitters in cortical afferent neurones, only dopamine is distributed to frontal and cingulate areas.34 All of the above argue for a possible role of dopamine in the neurotoxic effects of NMDA receptor antagonists. The data presented here (Fig. 5), showing that the dopamine D2 receptor antagonist haloperidol blocks the toxicity produced by both nitrous oxide and ketamine, provides supporting evidence for this view.
Possible clinical implications
Xenon, when used as a general anaesthetic, has virtually no side-effects28 29 and rapidly distributes into the CNS when breathed. Its ability to act as a neuroprotectant together with its apparent lack of neurotoxicity sets xenon apart from other anaesthetics, such as nitrous oxide and ketamine, with known NMDA antagonist properties. Thus, xenon might be considered as the anaesthetic of choice when neuroneal injury can be anticipated, for example in cardiac surgery requiring coronary artery bypass.35
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Acknowledgements |
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References |
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2 Choi DW, Koh JY, Peters S. Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists. J Neurosci 1988; 8: 18596[Abstract]
3 Sarraf-Yazdi S, Sheng H, Miura Y, et al. Relative neuroprotective effects of dizocilpine and isoflurane during focal cerebral ischemia in the rat. Anesth Analg 1998; 87: 728[Abstract]
4 Harada H, Kelly PJ, Cole DJ, Drummond JC, Patel PM. Isoflurane reduces N-methyl-D-aspartate toxicity in vivo in the rat cerebral cortex. Anesth Analg 1999; 89: 14427
5 Popovic R, Liniger R, Bickler PE. Anesthetics and mild hypothermia similarly prevent hippocampal neurone death in an in vitro model of cerebral ischemia. Anesthesiology 2000; 92: 13439[ISI][Medline]
6 Kudo M, Aono M, Lee Y, Massey G, Pearlstein RD, Warner DS. Effects of volatile anesthetics on N-methyl-D-aspartate excitotoxicity in primary rat neuroneal-glial cultures. Anesthesiology 2001; 95: 75665[ISI][Medline]
7 Arrowsmith JE, Harrison MJ, Newman SP, Stygall J, Timberlake N, Pugsley WB. Neuroprotection of the brain during cardiopulmonary bypass: a randomized trial of remacemide during coronary artery bypass in 171 patients. Stroke 1998; 29: 235762
8 Franks NP, Dickinson R, de Sousa SLM, Hall AC, Lieb WR. How does xenon produce anaesthesia? Nature 1998; 396: 324[ISI][Medline]
9 de Sousa SL, Dickinson R, Lieb WR, Franks NP. Contrasting synaptic actions of the inhalational general anesthetics isoflurane and xenon. Anesthesiology 2000; 92: 105566[ISI][Medline]
10 Anis NA, Berry SC, Burton NR, Lodge D. The dissociative anaesthetics, ketamine and phencyclidine, selectively reduce excitation of central mammalian neuronees by N-methyl-aspartate. Br J Pharmacol 1983; 79: 56575[Abstract]
11 Malhotra AK, Pinals DA, Weingartner H, et al. NMDA receptor function and human cognition: the effects of ketamine in healthy volunteers. Neuropsychopharmacology 1996; 14: 3017[ISI][Medline]
12 Olney JW, Labruyere J, Price MT. Pathological changes induced in cerebrocortical neurones by phencyclidine and related drugs. Science 1989; 244: 13602[ISI][Medline]
13 Allen HL, Iversen LL. Phencyclidine, dizocilpine, and cere brocortical neurones. Science 1990; 247: 221[ISI][Medline]
14 Jevtovic-Todorovic V, Todorovic SM, Mennerick S, et al. Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin. Nature Med 1998; 4: 4603[ISI][Medline]
15 Murakawa M, Adachi T, Nakao S, Seo N, Shingu K, Mori K. Activation of the cortical and medullary dopaminergic systems by nitrous oxide in rats: a possible neurochemical basis for psychotropic effects and postanesthetic nausea and vomiting. Anesth Analg 1994; 78: 37681[Abstract]
16 Moghaddam B, Adams B, Verma A, Daly D. Activation of glutamatergic neurotransmission by ketamine: a novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex. J Neurosci 1997; 17: 29217
17 Lindefors N, Barati S, OConnor WT. Differential effects of single and repeated ketamine administration on dopamine, serotonin and GABA transmission in rat medial prefrontal cortex. Brain Res 1997; 759: 20512[ISI][Medline]
18 Verma A, Moghaddam B. NMDA receptor antagonists impair prefrontal cortex function as assessed via spatial delayed alternation performance in rats: modulation by dopamine. J Neurosci 1996; 16: 3739[Abstract]
19 Petzelt CH. New concepts in neuroprotection. J Anästh Intensivbehandlung 2001; 3: S38
20 MacDonald MC, Robertson HA, Wilkinson M. Expression of c-fos protein by N-methyl-D-aspartic acid in hypothalamus of immature female rats: blockade by MK-801 or neonatal treatment with monosodium glutamate. Brain Res Dev 1990; 56: 2947[ISI][Medline]
21 Uemura Y, Kowall NW, Moskowitz MA. Focal ischemia in rats causes time-dependent expression of c-fos protein immunoreactivity in widespread regions of ipsilateral cortex. Brain Res 1991; 552: 99105[ISI][Medline]
22 Gass P, Herdegen T, Bravo R, Kiessling M. Induction and suppression of immediate early genes in specific rat brain regions by the non-competitive N-methyl-D-aspartate receptor antagonist MK-801. Neuroscience 1993; 53: 74958[ISI][Medline]
23 Walton M, MacGibbon G, Young D, et al. Do c-Jun, c-Fos, and amyloid precursor protein play a role in neuroneal death or survival? J Neurosci Res 1998; 53: 33042[ISI][Medline]
24 Zhai QZ, Traub RJ. The NMDA receptor antagonist MK-801 attenuates c-Fos expression in the lumbosacral spinal cord following repetitive noxious and non-noxious colorectal distention. Pain 1999; 83: 3219[ISI][Medline]
25 Jevtovic-Todorovic V, Wozniak DF, Benshoff ND, Olney JW. A comparative evaluation of the neurotoxic properties of ketamine and nitrous oxide. Brain Res 2001; 895: 2647[ISI][Medline]
26 Hasegawa K, Litt L, Espanol MT, Sharp FR, Chan PH. Expression of c-fos and hsp70 mRNA in neonatal rat cerebrocortical slices during NMDA-induced necrosis and apoptosis. Brain Res 1998; 785: 26278[ISI][Medline]
27 Sharp FR, Butman M, Aardalen K, et al. neuroneal injury produced by NMDA antagonists can be detected using heat shock proteins and can be blocked with antipsychotics. Psychopharmacol Bull 1994; 30: 55560[ISI][Medline]
28 Luttropp HH, Thomasson R, Dahm S, Persson J, Werner O. Clinical experience with minimal flow xenon anesthesia. Acta Anaesthesiol Scand 1994; 38: 1215[ISI][Medline]
29 Lynch C, 3rd, Baum J, Tenbrinck R. Xenon anesthesia. Anesthesiology 2000; 92: 8658[ISI][Medline]
30 Loscher W, Honack D. The behavioural effects of MK-801 in rats: involvement of dopaminergic, serotonergic and noradrenergic systems. Eur J Pharmacol 1992; 215: 199208[ISI][Medline]
31 Nishizawa N, Nakao S, Nagata A, Hirose T, Masuzawa M, Shingu K. The effect of ketamine isomers on both mice behavioral responses and c- Fos expression in the posterior cingulate and retrosplenial cortices. Brain Res 2000; 857: 18892[ISI][Medline]
32 Nagata A, Nakao Si S, Nishizawa N, et al. Xenon inhibits but N2O enhances ketamine-induced c-Fos expression in the rat posterior cingulate and retrosplenial cortices. Anesth Analg 2001; 92: 3628
33 Inomata S, Maze M, Hashimoto T, Jones M, Fujinaga M. Nitrous oxide induced met-enkephalin release provokes dopamine release in rat adrenal medulla cultured in vitro. Anesthesiology 2000; 93 (Suppl): A758
34 Jones EG. Neurotransmitters in the cerebral cortex. J Neurosurg 1986; 65: 13553[ISI][Medline]
35 Newman MF, Kirchner JL, Phillips-Bute B, et al. Longitudinal assessment of neurocognitive function after coronary-artery bypass surgery. N Engl J Med 2001; 344: 395402