Traumatic brain injury (TBI) is a leading cause of morbidity and mortality throughout the world. Despite improvements in medical care, the current clinical TBI treatment is mainly supportive and no specific neuroprotective drugs are available. TBI arises from external forces applied to the head, resulting in immediate and irreversible damage, or ‘primary injury’. In addition, early and long-lasting secondary injury cascades are triggered. Many of the debilitating functional impairments observed in patients result from the potentially preventable ‘secondary injury’. Over-activation of N-methyl-D-aspartate receptors is thought to play a key role in secondary injury. Xenon is a noble gas and general anaesthetic that is a competitive inhibitor of the N-methyl-D-aspartate receptor at the glycine binding site.1–3 Xenon has been shown to be neuroprotective in models of brain ischaemia. Much less is known about the xenon effect in the context of TBI. Our work focused on evaluating xenon’s neuroprotective efficacy in the reproducible controlled cortical impact animal model of blunt TBI, which includes elements found after moderate to severe TBI in humans, such as contusional lesion, brain oedema, elevated intracranial pressure, and neurological impairment. Adult C57BL/6 male mice (n=196) were fixed in a stereotactic frame under anaesthesia (sevoflurane 3.5% and buprenorphine s.c. 0.1 mg/kg) and underwent a right parietal cortical impact, delivered by a custom-made electropneumatic impactor with a 3-mm-diameter flat tip perpendicular to the brain surface. Impact velocity of 8 m s–1, impact duration of 150 ms, and brain-penetration depth of 1.0 mm were used. Throughout the procedure, the core body temperature was monitored and feedback controlled. The animals were randomly assigned to control (75% nitrogen:25% oxygen) and xenon treated (30%, 50%, or 75% xenon:25% oxygen, balanced with nitrogen) groups. Short-term and long-term outcomes, both functional and histological, were measured by researchers blinded to the treatment. The statistical significance was assessed with one-way and two-way analyses of variance with Bonferroni’s post hoc test. Our study showed 75% xenon significantly (P<0.05) reduced the contusion volume 24 h after injury, and significantly (P<0.05) improved the neurological outcome up to 4 days after injury and clinically relevant locomotor parameters 1 month after injury. Xenon treatment significantly (P<0.05) reduced the contusion volume when given up to 3 h after injury, and significantly (P<0.05) improved the neurological outcome when given up to 1 h after injury. Significant (P<0.05) reductions in the contusion volume and improvement in the neurological outcome 24 h after injury were also achieved with 30% and 50% xenon concentrations. Our results show in an animal model of TBI that xenon improves the functional outcomes and reduces the contusion volume. We demonstrated both a reduction in the development of secondary injury and an improvement in long-term translationally relevant motor outcomes. Our findings, including the demonstration of long-term neuroprotection and a clinically relevant therapeutic time window, support the idea that xenon may be of benefit as a neuroprotective treatment in TBI patients. 1.Armstrong SP, Banks P, McKitrick TJW, et al. Anesthesiology 2012; 117: 38–472.Dickinson R, Peterson BK, Banks P, et al. Anesthesiology 2007; 107: 756–673.Franks NP, Dickinson R, de Sousa SL, Hall AC, Lieb WR. Nature 1998; 396: 324

Background: Xenon, the inert anesthetic gas, is neuroprotective in models of brain injury. The authors investigate the neuroprotective mechanisms of the inert gases such as xenon, argon, krypton, neon, and helium in an in vitro model of traumatic brain injury.Methods: The authors use an in vitro model using mouse organotypic hippocampal brain slices, subjected to a focal mechanical trauma, with injury quantified by propidium iodide fluorescence. Patch clamp electrophysiology is used to investigate the effect of the inert gases on N-methyl-d-aspartate receptors and TREK-1 channels, two molecular targets likely to play a role in neuroprotection.Results: Xenon (50%) and, to a lesser extent, argon (50%) are neuroprotective against traumatic injury when applied after injury (xenon 431% protection at 72h after injury [N = 104]; argon 30 +/- 6% protection [N = 44]; mean +/- SEM). Helium, neon, and krypton are devoid of neuroprotective effect. Xenon (50%) prevents development of secondary injury up to 48h after trauma. Argon (50%) attenuates secondary injury, but is less effective than xenon (xenon 50 +/- 5% reduction in secondary injury at 72h after injury [N = 104]; argon 34 +/- 8% reduction [N = 44]; mean +/- SEM). Glycine reverses the neuroprotective effect of xenon, but not argon, consistent with competitive inhibition at the N-methyl-d-aspartate receptor glycine site mediating xenon neuroprotection against traumatic brain injury. Xenon inhibits N-methyl-d-aspartate receptors and activates TREK-1 channels, whereas argon, krypton, neon, and helium have no effect on these ion channels.Conclusions: Xenon neuroprotection against traumatic brain injury can be reversed by increasing the glycine concentration, consistent with inhibition at the N-methyl-d-aspartate receptor glycine site playing a significant role in xenon neuroprotection. Argon and xenon do not act via the same mechanism.

Objectives:To determine the neuroprotective efficacy of the inert gas xenon following traumatic brain injury and to determine whether application of xenon has a clinically relevant therapeutic time window. Design:Controlled animal study. Setting:University research laboratory. Subjects:Male C57BL/6N mice (n = 196). Interventions:Seventy-five percent xenon, 50% xenon, or 30% xenon, with 25% oxygen (balance nitrogen) treatment following mechanical brain lesion by controlled cortical impact. Measurements and Main Results:Outcome following trauma was measured using 1) functional neurologic outcome score, 2) histological measurement of contusion volume, and 3) analysis of locomotor function and gait. Our study shows that xenon treatment improves outcome following traumatic brain injury. Neurologic outcome scores were significantly (p < 0.05) better in xenon-treated groups in the early phase (24 hr) and up to 4 days after injury. Contusion volume was significantly (p < 0.05) reduced in the xenon-treated groups. Xenon treatment significantly (p < 0.05) reduced contusion volume when xenon was given 15 minutes after injury or when treatment was delayed 1 or 3 hours after injury. Neurologic outcome was significantly (p < 0.05) improved when xenon treatment was given 15 minutes or 1 hour after injury. Improvements in locomotor function (p < 0.05) were observed in the xenon-treated group, 1 month after trauma. Conclusions:These results show for the first time that xenon improves neurologic outcome and reduces contusion volume following traumatic brain injury in mice. In this model, xenon application has a therapeutic time window of up to at least 3 hours. These findings support the idea that xenon may be of benefit as a neuroprotective treatment in patients with brain trauma.

BACKGROUND:Xenon is a general anesthetic with neuroprotective properties. Xenon inhibition at the glycine-binding site of the N-Methyl-D-aspartate (NMDA) receptor mediates xenon neuroprotection against ischemic injury in vitro. Here we identify specific amino acids important for xenon binding to the NMDA receptor, with the aim of finding silent mutations that eliminate xenon binding but leave normal receptor function intact. METHODS:Site-directed mutagenesis was used to mutate specific amino-acids in the GluN1 subunit of rat NMDA receptors. Mutant GluN1/GluN2A receptors were expressed in HEK 293 cells and were assessed functionally using patch-clamp electrophysiology. The responses of the mutant receptors to glycine and anesthetics were determined. RESULTS:Mutation of phenylalanine 758 to an aromatic tryptophan or tyrosine left glycine affinity unchanged, but eliminated xenon binding without affecting the binding of sevoflurane or isoflurane. CONCLUSIONS:These findings confirm xenon binds to the glycine site of the GluN1 subunit of the NMDA receptor and indicate that interactions between xenon and the aromatic ring of the phenylalanine 758 residue are important for xenon binding. Our most important finding is that we have identified two mutations, F758W and F758Y, that eliminate xenon binding to the NMDA receptor glycine site without changing the glycine affinity of the receptor or the binding of volatile anesthetics. The identification of these selective mutations will allow knock-in animals to be used to dissect the mechanism(s) of xenon's neuroprotective and anesthetic properties in vivo.