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Pain Medicine  |   June 2002
Effects of Xenon on In Vitro  and In Vivo  Models of Neuronal Injury
Author Affiliations & Notes
  • Stefan Wilhelm, M.D.
    *
  • Daqing Ma, M.D., Ph.D.
    *
  • Mervyn Maze, M.B., Ch.B., F.R.C.P., F.R.C.A.
  • Nicholas P. Franks, Ph.D.
  • *Postdoctoral Research Fellow, Chelsea & Westminster Hospital. †Sir Ivan Magill Professor of Anaesthetics, ‡Professor of Biophysics and Anaesthetics, Chelsea & Westminster Hospital, and the Biophysics Group, The Blackett Laboratory, Imperial College of Science, Technology and Medicine.
  • Received from the Magill Department of Anaesthesia, Chelsea & Westminster Hospital, and the Biophysics Group, The Blackett Laboratory, Imperial College of Science, Technology and Medicine, London, United Kingdom.
Article Information
Pain Medicine
Pain Medicine   |   June 2002
Effects of Xenon on In Vitro  and In Vivo  Models of Neuronal Injury
Anesthesiology 6 2002, Vol.96, 1485-1491. doi:
Anesthesiology 6 2002, Vol.96, 1485-1491. doi:
IN marked contrast to most general anesthetic agents, 1,2 xenon, at surgically relevant concentrations, produces little or no potentiation at γ-aminobutyric acid synapses 3,4 or on γ-aminobutyric acid type A receptors in in vitro  expression systems. 5,6 Rather, is an effective inhibitor of glutamatergic N  -methyl-d-aspartate (NMDA) receptors, yet an apparently weak inhibitor of the non-NMDA subtypes of glutamate receptors. 3,4 
The NMDA subtype of the glutamate receptor is a target for many putative neuroprotective agents because its activation is required to sustain ongoing neuronal injury and death. 7 Notwithstanding their putative beneficial effect in animal models, the development of several NMDA receptor antagonists has had to be abandoned because clinical trials revealed that they possessed their own inherent neurotoxicity (characterized by a specific lesion in the retrosplenial cortex) or because of their failure to reach the site of injury. 8 Previous studies revealed that xenon, uniquely among anesthetics with NMDA receptor antagonist properties, does not appear to produce neurotoxicity in the retrosplenial cortex; rather, xenon protects against the neurotoxic injury produced by ketamine. 9 Furthermore, xenon readily partitions across the blood–brain barrier and can be expected to access the site of central nervous system injury rapidly.
In a series of in vitro  and in vivo  models, we determined whether xenon protects against neuronal injury. The results demonstrate that xenon exerts potent neuroprotection at subanesthetic concentrations in each of the models examined.
Materials and Methods
This study conformed to the United Kingdom Animals (Scientific Procedures) Act of 1986.
In Vitro  Experiments
Cell Culture.
Mixed cortical cell cultures, containing both neuronal and glial elements, were prepared using the methods described previously 10,11 with minor modifications. Briefly, whole cerebral neocortices (devoid of the hippocampal formation and basal ganglia) were removed from fetal BALB/c mice at 14–16 days’ gestation. After trypsination and resuspension, cells were plated on a confluent monolayer of astrocytes at a density of 1–1.25 × 105cells/cm2on 24-multiwell plates (Costar, Cambridge, MA). Cortical glial cell cultures were prepared from early postnatal BALB/c mice (postnatal days 1–3) and reached confluence within approximately 1 week after plating. The mixed glial–neuronal cocultures were used at 15 ± 1 days.
Exposure to Excitatory Amino Acids.
For the experiments in which either NMDA or glutamate was exogenously applied, the culture medium had been carefully replaced by HEPES buffer (120 mm NaCl, 5.4 mm KCl, 0.8 mm MgCl2, 1.8 mm CaCl2, 15 mm glucose, and 20 mm HEPES, titrated to pH 7.4 using 1 m NaOH). Agonist exposure was generally performed at room temperature in room air (22 ± 2°C). After 10-min exposure, NMDA or glutamate was washed out, and culture medium free of glutamate and lactate dehydrogenase (LDH; Eagle minimum essential medium augmented with 25 mm glucose and 38 mm bicarbonate) was maintained until the end of the experiment.
Preparation of Xenon Solutions.
Solutions for the xenon experiments were prepared by first bubbling pure gases (oxygen, nitrogen, or xenon) through fine sintered glass bubblers in 250- or 500-ml Drechsel bottles filled with the above-described HEPES buffer. Solutions were bubbled for 20 min (in preliminary experiments, the xenon concentration was monitored using gas chromatography to confirm that this time was sufficient for equilibration). To minimize oxidation, the excitatory amino acids were absent in the fully oxygenated buffer but present at the appropriate concentrations in the xenon and nitrogen solutions. During bubbling, the solutions were continually stirred at room temperature. As a final step, these solutions were mixed to achieve the desired final concentrations of the respective gases. Immediately before the experiment, NMDA or glutamate was added to 15 ml of the gas-containing solutions and carefully mixed to obtain the desired NMDA and glutamate concentrations. Preliminary experiments using a dye indicated that good mixing was achieved.
Gas Exposure Chambers.
To effect continuous delivery of xenon, a purpose-built, airtight, temperature-controlled cell-culture chamber, equipped with inlet and outlet valves and an internal electric fan, was constructed. When connected to calibrated flowmeters for xenon, oxygen, nitrogen, and carbon dioxide, the desired gas composition could be obtained. In all experiments, 20% oxygen and 5% carbon dioxide were used, with the remainder being xenon or nitrogen in various proportions. Total gas flow was 100 ml/min, and the chamber was flushed for 30 min before establishing a closed system. With this protocol, the final gas concentrations (determined using gas chromatography) were found to be stable, and there was no measurable leakage over 24 h.
Oxygen Deprivation.
Oxygen deprivation was performed as described previously 12 with minor modifications. Briefly, the culture medium was replaced with prewarmed (37°C) deoxygenated balanced salt solution, pH 7.4 (NaCl 116 mm, KCl 5.4 mm, MgSO40.8 mm, NaH2PO41.0 mm, CaCl21.8 mm, NaHCO326 mm, glucose 5 mm, pH adjustment with 2 m HCl). Immediately after replacement of the medium, the cell cultures were transferred into the gas exposure chamber (containing 95% nitrogen, 5% carbon dioxide) for 75 min at 37°C. Oxygen deprivation was terminated by exchanging oxygenated minimum essential medium before the cultures were returned to a normoxic incubator (95% air, 5% carbon dioxide,) or into the xenon-containing gas exposure chamber (20% oxygen, 5% carbon dioxide, with the remainder being chosen concentrations of xenon and nitrogen) for the subsequent 24 h at 37°C.
Assessment of Neuronal Injury.
The neuronal injury was quantified by the amount of LDH released into the medium over the appropriate time period. As previously reported, 13 this convenient and quantitative index correlated with morphologic estimate and was used as the index of injury. LDH activity in each sample was determined in triplicate by spectrophotometric measurements (Shimadzu UV 1201 photometer; Milton Keynes, United Kingdom) at room temperature using a standardized colorimetric enzyme kit (Sigma 500 C; Sigma-Aldrich, Poole, United Kingdom). The amount of LDH released by sham-washed controls was subtracted to give the LDH signal specific to the injury, neuronal protection was expressed as a percentage of the maximal LDH released, and the IC50concentration was defined as the xenon concentration that reduced the release to 50% of the control value.
In Vivo  Experiments
Four groups of female Sprague-Dawley rats (weight, 240–260 g) were treated as follows: group 1 (control, n = 6) received 0.8 ml/100 g saline subcutaneously; group 2 (n = 7) received 100 mg/kg N  -methyl-dl-aspartate (NMA) subcutaneously; group 3 (n = 5) received 75% xenon in oxygen; group 4 was subdivided into four treatment subgroups and exposed to 20% xenon (n = 5), 40% xenon (n = 5), 60% xenon (n = 5), or 75% xenon (n = 8), with the remainder being nitrogen in various proportions and 25% oxygen for 15 min before subcutaneous injection of 100 mg/kg NMA. The dose of NMA was selected on the basis of a previous study, 15 and it produced no obvious behavioral effects. Similarly, xenon induced no behavioral effects, either on its own or after NMA injection, with the exception of a mild and short-lived (1–2 min) period of motor hyperexcitability that was observed at the highest concentration used (75% xenon).
During exposure to 20, 40, 60, and 75% xenon (as previously mentioned), the gas mixture was introduced into a chamber (International Market Supply, Cheshire, United Kingdom) using calibrated flowmeters. After a 3-min flush at a flow rate of 4 l/min, the flow rate was reduced to 40 ml/min for the remaining 3 h. The carbon dioxide level was kept less than 0.6% with soda lime, and the humidity was kept between 40 and 60% using silica gel (Merck, Leicestershire, United Kingdom).
Histology.
Three hours after treatment was initiated, animals were killed by an overdose of pentobarbital, perfused transcardially with 4% paraformaldehyde in 0.1 m phosphate buffer, and the whole brain was removed, sliced, and embedded in paraffin. Three sections (4 μm) cut midway between the rostral and caudal boundaries of the arcuate nucleus were stained with cresyl violet for light microscopic evaluation. To determine the amount of neuronal damage in the arcuate nucleus, degenerated neurons were identified by their pyknotic nuclei and edematous swelling of the surrounding cytoplasmic compartment, giving the appearance of a bull's eye. The sum of degenerated neurons of three representative sections was used.
To determine whether xenon induces toxicity, layers three and four of the retrosplenial cortex were examined for intracytoplasmic vacuolization using the criteria established by Fix et al.  14 The sum of vacuolated neurons of six snapshots (acquired by Zeiss AxioCam Digital Camera; Zeiss, Welwyn Garden City, United Kingdom) of the retrosplenial cortex from each of four rats was used.
Reagents and Solutions
Minimal essential medium, fetal bovine serum, horse serum, and murine epidermal growth factor were obtained from GibcoBRL, Life Technologies Ltd. (Paisley, United Kingdom). NMDA, NMA, glutamate, and the colorimetric kit for the determination of LDH were purchased from Sigma-Aldrich (Poole, United Kingdom). Unless otherwise stated, all other chemicals were obtained from Merck (Leicestershire, United Kingdom). Beside xenon, which was obtained from Air Products (Crewe, United Kingdom), all other gases were supplied by BOC Gases (Guildford, Surrey, United Kingdom).
Data Analysis
In vitro  data were tested using analysis of variance with post hoc  t  test and correction for multiple comparisons using Bonferroni correction. For the in vivo  data, statistical analysis was performed by analysis of variance followed by the Newman-Keuls test.
Results
In control experiments, brief (10 min) exposures of the cell cultures to either NMDA (fig. 1A) or glutamate (fig. 1B) resulted in a concentration-dependent increase in the LDH released into the culture medium. Microscopic examination of the cell cocultures showed widespread neuronal disintegration, whereas the morphology of the underlying astroglia bed appeared relatively unaffected (not shown). The NMDA- or glutamate-induced LDH release was markedly larger than baseline release (i.e.  , in the absence of added NMDA or glutamate) and saturated at NMDA and glutamate concentrations in excess of approximately 100 μm. The EC50concentrations for NMDA- and glutamate-induced injury were 46 ± 8 and 66 ± 19 μm, respectively (mean ± SEM). LDH assays performed at times between 6 and 24 h (fig. 1C) showed a similar degree of injury.
Fig. 1. Concentration–response relation for N  -methyl-d-aspartate (NMDA) and glutamate neurotoxicity and time course for injury. Cortical neuronal cultures were exposed for 10 min to either NMDA (A  ) or glutamate (B  ), and injury was quantified by the amount of lactate dehydrogenase (LDH) released into the bathing medium after 24 h. The EC50concentrations for NMDA- and glutamate-induced injury were 46 ± 8 and 66 ± 19 μm, respectively (mean ± SEM). (C  ) Neuronal damage provoked by 250 μm NMDA (squares) or 100 μm glutamate (circles) was essentially complete 6 h after injury and did not change greatly up to 24 h after injury. Baseline LDH release was typically about 50 units. Data are presented as mean ± SEM (n = 3).
Fig. 1. Concentration–response relation for N 
	-methyl-d-aspartate (NMDA) and glutamate neurotoxicity and time course for injury. Cortical neuronal cultures were exposed for 10 min to either NMDA (A 
	) or glutamate (B 
	), and injury was quantified by the amount of lactate dehydrogenase (LDH) released into the bathing medium after 24 h. The EC50concentrations for NMDA- and glutamate-induced injury were 46 ± 8 and 66 ± 19 μm, respectively (mean ± SEM). (C 
	) Neuronal damage provoked by 250 μm NMDA (squares) or 100 μm glutamate (circles) was essentially complete 6 h after injury and did not change greatly up to 24 h after injury. Baseline LDH release was typically about 50 units. Data are presented as mean ± SEM (n = 3).
Fig. 1. Concentration–response relation for N  -methyl-d-aspartate (NMDA) and glutamate neurotoxicity and time course for injury. Cortical neuronal cultures were exposed for 10 min to either NMDA (A  ) or glutamate (B  ), and injury was quantified by the amount of lactate dehydrogenase (LDH) released into the bathing medium after 24 h. The EC50concentrations for NMDA- and glutamate-induced injury were 46 ± 8 and 66 ± 19 μm, respectively (mean ± SEM). (C  ) Neuronal damage provoked by 250 μm NMDA (squares) or 100 μm glutamate (circles) was essentially complete 6 h after injury and did not change greatly up to 24 h after injury. Baseline LDH release was typically about 50 units. Data are presented as mean ± SEM (n = 3).
×
To assess the possible neuroprotective effects of xenon, LDH assays were performed 6 h after brief exposures to either NMDA or glutamate (at, or close to, concentrations that induced maximal LDH release) in the presence of increasing concentrations of xenon. LDH release was significantly reduced at all concentrations tested (fig. 2), with xenon IC50concentrations for neuroprotection being 19 ± 6% and 28 ± 8% for NMDA and glutamate-induced injury, respectively (mean ± SEM). Xenon at 75% atm had no significant effect (P  > 0.7) on baseline LDH release (i.e.  , in the absence of added NMDA or glutamate), indicating no intrinsic neurotoxicity of xenon alone. Xenon was also effective (IC50= 10 ± 4% atm) in protecting against the injury caused by depriving the cell cultures of oxygen (fig. 2C) for 75 min.
Fig. 2. Xenon exerts a concentration-dependent neuroprotective effect in vitro  . Neuronal cultures were treated with either (A  ) 250 μm NMDA or (B  ) 100 μm glutamate for 10 min in the presence of increasing concentrations of xenon. The xenon IC50concentrations for neuroprotection were 19 ± 6 and 28 ± 8% for NMDA and glutamate-induced injury, respectively (mean ± SEM). (C  ) Xenon was also neuroprotective in cultures deprived of oxygen (circles) for 75 min before rescue with medium containing 20% oxygen. In all cases, neuronal injury is expressed as a percentage of the maximum lactate dehydrogenase (LDH) release after 6 h determined in the absence of xenon. The points represent mean ± SEM (n = 3).
Fig. 2. Xenon exerts a concentration-dependent neuroprotective effect in vitro 
	. Neuronal cultures were treated with either (A 
	) 250 μm NMDA or (B 
	) 100 μm glutamate for 10 min in the presence of increasing concentrations of xenon. The xenon IC50concentrations for neuroprotection were 19 ± 6 and 28 ± 8% for NMDA and glutamate-induced injury, respectively (mean ± SEM). (C 
	) Xenon was also neuroprotective in cultures deprived of oxygen (circles) for 75 min before rescue with medium containing 20% oxygen. In all cases, neuronal injury is expressed as a percentage of the maximum lactate dehydrogenase (LDH) release after 6 h determined in the absence of xenon. The points represent mean ± SEM (n = 3).
Fig. 2. Xenon exerts a concentration-dependent neuroprotective effect in vitro  . Neuronal cultures were treated with either (A  ) 250 μm NMDA or (B  ) 100 μm glutamate for 10 min in the presence of increasing concentrations of xenon. The xenon IC50concentrations for neuroprotection were 19 ± 6 and 28 ± 8% for NMDA and glutamate-induced injury, respectively (mean ± SEM). (C  ) Xenon was also neuroprotective in cultures deprived of oxygen (circles) for 75 min before rescue with medium containing 20% oxygen. In all cases, neuronal injury is expressed as a percentage of the maximum lactate dehydrogenase (LDH) release after 6 h determined in the absence of xenon. The points represent mean ± SEM (n = 3).
×
We went on to investigate whether the neuroprotection that was evident in vitro  could be observed in vivo  . We used a well-established in vivo  model of neurotoxicity that involved the subcutaneous injection of NMA and the subsequent neuronal degeneration that occurs in the arcuate nucleus of the hypothalamus. 15 No degenerated neurons were found in either control animals or in those tested with xenon alone. We confirmed that characteristic and reproducible neuronal degeneration occurs after NMA injection that is easily distinguishable from sham-injected animals (figs. 3A and B). Figure 3Cshows that, in the presence of 75% xenon, the number of degenerated neurons (characterized by cytoplasmic swelling and pyknotic nuclei) is greatly reduced. We quantified the extent of neuroprotection by counting the number of degenerated neurons after the animals had been exposed to increasing concentrations of xenon. The number of degenerated neurons decreased with increasing xenon concentration, with an IC50concentration of 78 ± 8% (mean ± SEM;fig. 3D).
Fig. 3. Xenon exerts a concentration-dependent neuroprotective effect in vivo  . Coronal sections containing the arcuate nucleus of the hypothalamus were stained with cresyl violet and visualized at a magnification of 200 times. A control section is shown (A  ) where the scale bar represents 20 μm. Subcutaneous injection of 100 mg/kg N  -methyl-dl-aspartate induced a characteristic neurodegeneration in the arcuate nucleus of the hypothalamus (B  ), and this was greatly reduced in the presence of 75% xenon (C  ). The neuroprotective effect of xenon was concentration-dependent (D  ) and was quantified by counting the number of degenerated neurons in both arcuate nuclei over a range of xenon concentrations. At greater than 40% xenon, the number of degenerated neurons was significantly reduced at the 5% level (*) and at and above 60% xenon, protection was significant at the 1% level (**). The points represent mean ± SEM (n = 5–8).
Fig. 3. Xenon exerts a concentration-dependent neuroprotective effect in vivo 
	. Coronal sections containing the arcuate nucleus of the hypothalamus were stained with cresyl violet and visualized at a magnification of 200 times. A control section is shown (A 
	) where the scale bar represents 20 μm. Subcutaneous injection of 100 mg/kg N 
	-methyl-dl-aspartate induced a characteristic neurodegeneration in the arcuate nucleus of the hypothalamus (B 
	), and this was greatly reduced in the presence of 75% xenon (C 
	). The neuroprotective effect of xenon was concentration-dependent (D 
	) and was quantified by counting the number of degenerated neurons in both arcuate nuclei over a range of xenon concentrations. At greater than 40% xenon, the number of degenerated neurons was significantly reduced at the 5% level (*) and at and above 60% xenon, protection was significant at the 1% level (**). The points represent mean ± SEM (n = 5–8).
Fig. 3. Xenon exerts a concentration-dependent neuroprotective effect in vivo  . Coronal sections containing the arcuate nucleus of the hypothalamus were stained with cresyl violet and visualized at a magnification of 200 times. A control section is shown (A  ) where the scale bar represents 20 μm. Subcutaneous injection of 100 mg/kg N  -methyl-dl-aspartate induced a characteristic neurodegeneration in the arcuate nucleus of the hypothalamus (B  ), and this was greatly reduced in the presence of 75% xenon (C  ). The neuroprotective effect of xenon was concentration-dependent (D  ) and was quantified by counting the number of degenerated neurons in both arcuate nuclei over a range of xenon concentrations. At greater than 40% xenon, the number of degenerated neurons was significantly reduced at the 5% level (*) and at and above 60% xenon, protection was significant at the 1% level (**). The points represent mean ± SEM (n = 5–8).
×
We observed no significant change in the percentage of neurons in the retrosplenial cortex showing intracytoplasmic vacuolations in rats treated with 75% xenon (3.4 ± 0.7%; mean ± SD) when compared with rats administered normal saline (4.1 ± 1.1%; mean ± SD).
Discussion
Using a primary culture of neuronal and glial cells from the cerebral cortex of neonatal mice, predictable neuronal injury (as reflected by the release of LDH into the culture medium) is produced by NMDA, glutamate, and oxygen deprivation, as reported previously (fig. 1). 10–12,16 Xenon, in a concentration-dependent manner, reduced neuronal injury produced by each of NMDA (fig. 2A), glutamate (fig. 2B), and oxygen deprivation (fig. 2C). Neuronal protection was complete for the injury produced by oxygen-deprivation studies and up to 80% complete for the injury produced by each of NMDA and glutamate. In a validated in vivo  model of brain injury in rats, 15 xenon, in a concentration-dependent manner, reduced neuronal degeneration in the arcuate nucleus of the hypothalamus provoked by NMA (fig. 3). In each of the in vitro  and in vivo  paradigms, xenon exerted its neuroprotective effect at subanesthetic concentrations. 17,18 This might be clinically relevant because other anesthetics with known neuroprotective properties, such as propofol or pentobarbital, need to be administered at far higher concentrations to mitigate oxidative stress in primary astrocyte cultures 19 or to protect against ischemic brain injury in animal models. 20,21 
Xenon has been in anesthetic use for more than 50 years and has been demonstrated to have a remarkably safe clinical profile. 18 It is regarded as an anesthetic agent with minimal cardiorespiratory and renal effects. 22,23 A major obstacle to its widespread clinical application is its scarcity (representing no more than 0.00000875% of the atmosphere) and, as a consequence, the cost associated with its extraction. Therefore, it is anticipated that the use of xenon as a clinical anesthetic will be confined to settings in which cost–benefit analysis justifies its application.
In each of the experimental paradigms, xenon was administered before the injury was provoked; therefore, if they are able to be extrapolated to clinical settings, these data should be considered analogous to preemptive therapy in anticipation of a forthcoming neurologic insult (such as cardiopulmonary bypass or surgery for intracranial arteriovenous malformations or neoplasms). Although each of the models that were used has been validated in previous studies, 10,12,15 it must be recognized that these models may not necessarily be the neuropathologic correlates of disease processes involved in ischemia or trauma. It is also possible that the neuroprotective effect of xenon may not necessarily be caused by its well-defined antagonism 3,4 of the NMDA receptor;e.g.  , it is theoretically possible that the in vivo  neuroprotective effect may be caused by a decrease in brain temperature that was not monitored.
Previously, several NMDA receptor antagonists have shown remarkable efficacy against neurologic injury in preclinical models but failed to live up to their promise when subsequently investigated in clinical settings. 7,24 There are several possible reasons for the ineffective clinical translation of NMDA receptor antagonists. First, many of the NMDA receptor antagonists do not readily penetrate the blood–brain barrier, requiring large doses to be administered systemically; this, in turn, can produce systemic toxicity. 24 Others have been shown to block certain serotonin receptors and calcium channels in addition to NMDA receptors, which limits their clinical usefulness. 25 It is notable that xenon, a small uncharged atom, readily crosses the blood–brain barrier, producing a rapid onset of central nervous system effects in humans within the first minute of administration. Even for NMDA antagonists that do traverse the blood–brain barrier, central nervous system neurotoxicity may evolve. 24 Xenon is devoid of intrinsic neurotoxicity, as evidenced by the lack of vacuolization in layers three and four of the retrosplenial cortex, a region particularly susceptible to injury by NMDA antagonists. 14 In fact, xenon itself may prevent the toxicity associated with other NMDA antagonists. 9 Of course, one cannot discount the possibility that the reason for the clinical failures of NMDA antagonists may be that blockade of this glutamate receptor subtype may be insufficient to protect against injury.
Apart from anesthetics with NMDA receptor antagonist properties, other anesthetics have also been tested for their ability to mitigate neuronal injury in various paradigms. 19–21,26–28 Recently, Kudo et al.  28 examined the neuroprotective effects of several volatile anesthetic agents in a similar in vitro  model of NMDA excitotoxicity. Remarkably, in the clinical anesthetic range (i.e.  , approximately 1 minimum alveolar concentration), the neuroprotective effect was only approximately 20%, which is more than fourfold lower than both their observation with MK-801 and our observations with xenon. However, it is notable that volatile anesthetics may exert a more potent effect in vivo  than is seen in vitro  , 27 possibly because of the additional neuroprotective effect that activation of γ-aminobutyric acid type A receptors may confer. 29 In an in vivo  experimental paradigm, which is roughly comparable to that used by Harada et al.  , 27 xenon does appear to be a more potent neuroprotectant (fig. 3) than isoflurane.
Xenon may represent a useful neuroprotectant for preemptive use in clinical settings, in which NMDA receptor activation is pivotal to the production of injury, because of its known lack of toxic side effects and the relative ease with which it can be delivered to the brain. We are now embarking on clinical trials to investigate whether cognitive deficits, which develop in patients undergoing cardiopulmonary bypass in the course of their cardiac surgery, 30,31 can be prevented by administering xenon as part of the anesthetic regimen.
The authors thank Vesna Jevtovic-Todorovic, M.D., Ph.D. (Department of Anesthesiology, Washington University, St. Louis, MO), for helpful advice on the histological procedures.
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Fig. 1. Concentration–response relation for N  -methyl-d-aspartate (NMDA) and glutamate neurotoxicity and time course for injury. Cortical neuronal cultures were exposed for 10 min to either NMDA (A  ) or glutamate (B  ), and injury was quantified by the amount of lactate dehydrogenase (LDH) released into the bathing medium after 24 h. The EC50concentrations for NMDA- and glutamate-induced injury were 46 ± 8 and 66 ± 19 μm, respectively (mean ± SEM). (C  ) Neuronal damage provoked by 250 μm NMDA (squares) or 100 μm glutamate (circles) was essentially complete 6 h after injury and did not change greatly up to 24 h after injury. Baseline LDH release was typically about 50 units. Data are presented as mean ± SEM (n = 3).
Fig. 1. Concentration–response relation for N 
	-methyl-d-aspartate (NMDA) and glutamate neurotoxicity and time course for injury. Cortical neuronal cultures were exposed for 10 min to either NMDA (A 
	) or glutamate (B 
	), and injury was quantified by the amount of lactate dehydrogenase (LDH) released into the bathing medium after 24 h. The EC50concentrations for NMDA- and glutamate-induced injury were 46 ± 8 and 66 ± 19 μm, respectively (mean ± SEM). (C 
	) Neuronal damage provoked by 250 μm NMDA (squares) or 100 μm glutamate (circles) was essentially complete 6 h after injury and did not change greatly up to 24 h after injury. Baseline LDH release was typically about 50 units. Data are presented as mean ± SEM (n = 3).
Fig. 1. Concentration–response relation for N  -methyl-d-aspartate (NMDA) and glutamate neurotoxicity and time course for injury. Cortical neuronal cultures were exposed for 10 min to either NMDA (A  ) or glutamate (B  ), and injury was quantified by the amount of lactate dehydrogenase (LDH) released into the bathing medium after 24 h. The EC50concentrations for NMDA- and glutamate-induced injury were 46 ± 8 and 66 ± 19 μm, respectively (mean ± SEM). (C  ) Neuronal damage provoked by 250 μm NMDA (squares) or 100 μm glutamate (circles) was essentially complete 6 h after injury and did not change greatly up to 24 h after injury. Baseline LDH release was typically about 50 units. Data are presented as mean ± SEM (n = 3).
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Fig. 2. Xenon exerts a concentration-dependent neuroprotective effect in vitro  . Neuronal cultures were treated with either (A  ) 250 μm NMDA or (B  ) 100 μm glutamate for 10 min in the presence of increasing concentrations of xenon. The xenon IC50concentrations for neuroprotection were 19 ± 6 and 28 ± 8% for NMDA and glutamate-induced injury, respectively (mean ± SEM). (C  ) Xenon was also neuroprotective in cultures deprived of oxygen (circles) for 75 min before rescue with medium containing 20% oxygen. In all cases, neuronal injury is expressed as a percentage of the maximum lactate dehydrogenase (LDH) release after 6 h determined in the absence of xenon. The points represent mean ± SEM (n = 3).
Fig. 2. Xenon exerts a concentration-dependent neuroprotective effect in vitro 
	. Neuronal cultures were treated with either (A 
	) 250 μm NMDA or (B 
	) 100 μm glutamate for 10 min in the presence of increasing concentrations of xenon. The xenon IC50concentrations for neuroprotection were 19 ± 6 and 28 ± 8% for NMDA and glutamate-induced injury, respectively (mean ± SEM). (C 
	) Xenon was also neuroprotective in cultures deprived of oxygen (circles) for 75 min before rescue with medium containing 20% oxygen. In all cases, neuronal injury is expressed as a percentage of the maximum lactate dehydrogenase (LDH) release after 6 h determined in the absence of xenon. The points represent mean ± SEM (n = 3).
Fig. 2. Xenon exerts a concentration-dependent neuroprotective effect in vitro  . Neuronal cultures were treated with either (A  ) 250 μm NMDA or (B  ) 100 μm glutamate for 10 min in the presence of increasing concentrations of xenon. The xenon IC50concentrations for neuroprotection were 19 ± 6 and 28 ± 8% for NMDA and glutamate-induced injury, respectively (mean ± SEM). (C  ) Xenon was also neuroprotective in cultures deprived of oxygen (circles) for 75 min before rescue with medium containing 20% oxygen. In all cases, neuronal injury is expressed as a percentage of the maximum lactate dehydrogenase (LDH) release after 6 h determined in the absence of xenon. The points represent mean ± SEM (n = 3).
×
Fig. 3. Xenon exerts a concentration-dependent neuroprotective effect in vivo  . Coronal sections containing the arcuate nucleus of the hypothalamus were stained with cresyl violet and visualized at a magnification of 200 times. A control section is shown (A  ) where the scale bar represents 20 μm. Subcutaneous injection of 100 mg/kg N  -methyl-dl-aspartate induced a characteristic neurodegeneration in the arcuate nucleus of the hypothalamus (B  ), and this was greatly reduced in the presence of 75% xenon (C  ). The neuroprotective effect of xenon was concentration-dependent (D  ) and was quantified by counting the number of degenerated neurons in both arcuate nuclei over a range of xenon concentrations. At greater than 40% xenon, the number of degenerated neurons was significantly reduced at the 5% level (*) and at and above 60% xenon, protection was significant at the 1% level (**). The points represent mean ± SEM (n = 5–8).
Fig. 3. Xenon exerts a concentration-dependent neuroprotective effect in vivo 
	. Coronal sections containing the arcuate nucleus of the hypothalamus were stained with cresyl violet and visualized at a magnification of 200 times. A control section is shown (A 
	) where the scale bar represents 20 μm. Subcutaneous injection of 100 mg/kg N 
	-methyl-dl-aspartate induced a characteristic neurodegeneration in the arcuate nucleus of the hypothalamus (B 
	), and this was greatly reduced in the presence of 75% xenon (C 
	). The neuroprotective effect of xenon was concentration-dependent (D 
	) and was quantified by counting the number of degenerated neurons in both arcuate nuclei over a range of xenon concentrations. At greater than 40% xenon, the number of degenerated neurons was significantly reduced at the 5% level (*) and at and above 60% xenon, protection was significant at the 1% level (**). The points represent mean ± SEM (n = 5–8).
Fig. 3. Xenon exerts a concentration-dependent neuroprotective effect in vivo  . Coronal sections containing the arcuate nucleus of the hypothalamus were stained with cresyl violet and visualized at a magnification of 200 times. A control section is shown (A  ) where the scale bar represents 20 μm. Subcutaneous injection of 100 mg/kg N  -methyl-dl-aspartate induced a characteristic neurodegeneration in the arcuate nucleus of the hypothalamus (B  ), and this was greatly reduced in the presence of 75% xenon (C  ). The neuroprotective effect of xenon was concentration-dependent (D  ) and was quantified by counting the number of degenerated neurons in both arcuate nuclei over a range of xenon concentrations. At greater than 40% xenon, the number of degenerated neurons was significantly reduced at the 5% level (*) and at and above 60% xenon, protection was significant at the 1% level (**). The points represent mean ± SEM (n = 5–8).
×