Xenon improves neurologic outcome and reduces secondary injury following trauma in an in vivo model of traumatic brain injury.

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.

Neuroprotection against traumatic brain injury by xenon, but not argon, is mediated by inhibition at the N-methyl-D-aspartate receptor glycine site.

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 43±1% protection at 72 h 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 48 h after trauma. Argon (50%) attenuates secondary injury, but is less effective than xenon (xenon 50±5% reduction in secondary injury at 72 h 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.

Identification of two mutations (F758W and F758Y) in the N-methyl-D-aspartate receptor glycine-binding site that selectively prevent competitive inhibition by xenon without affecting glycine binding.

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.