Anesthesiology 2007; 106:746 –53 Copyright © 2007, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Xenon Mitigates Isoflurane-induced Neuronal Apoptosis in the Developing Rodent Brain Daqing Ma, M.D., Ph.D.,* Peter Williamson, M.B., B.S.,† Adam Januszewski, B.Sc.,‡ Marie-Caroline Nogaro, B.Sc.,‡ Mahmuda Hossain, Ph.D.,§ Lay Ping Ong,‡ Yi Shu, BSc., Nicholas P. Franks, Ph.D., F.Med.Sci.,# Mervyn Maze, M.B., Ch.B., F.Med.Sci.** Background: Anesthetics, including isoflurane and nitrous oxide, an antagonist of the N-methyl-D-aspartate subtype of the glutamate receptor, have been demonstrated to induce apopto- tic neurodegeneration when administered during neurodevel- opment. Xenon, also an N-methyl-D-aspartate antagonist, not only lacks the characteristic toxicity produced by other N-meth- yl-D-aspartate antagonists, but also attenuates the neurotoxicity produced by this class of agent. Therefore, the current study sought to investigate xenon’s putative protective properties against anesthetic-induced neuronal apoptosis. Method: Separate cohorts (n  5 or 6 per group) of 7-day-old rats were randomly assigned and exposed to eight gas mixtures: air, 75% nitrous oxide, 75% xenon, 0.75% isoflurane, 0.75% isoflurane plus 35% or 75% nitrous oxide, 0.75% isoflurane plus 30% or 60% xenon for 6 h. Rats were killed, and cortical and hippocampal apoptosis was assessed using caspase-3 immuno- staining. In separate cohorts, cortices were isolated for immuno- blotting of caspase 3, caspase 8, caspase 9, and cytochrome c. Organotypic hippocampal slices of postnatal mice pups were de- rived and cultured for 24 h before similar gas exposures, as above, and subsequently processed for caspase-3 immunostaining. Results: In vivo administration of isoflurane enhances neu- ronal apoptosis. When combined with isoflurane, nitrous oxide significantly increases whereas xenon significantly reduces ap- optosis to a value no different from that of controls. In vitro studies corroborate the ability of xenon to attenuate isoflurane- induced apoptosis. Isoflurane enhanced expression of indica- tors of the intrinsic and common apoptotic pathways; this en- hancement was increased by nitrous oxide but attenuated by xenon. Conclusions: The current study demonstrates that xenon pre- vents isoflurane-induced neonatal neuronal apoptosis. SYNAPTOGENESIS is a highly regulated period of brain development that is exquisitely sensitive to environmen- tal influences. The processes involved in neurodevelop- ment are conserved among species; in rodents, synapto- genesis occurs predominantly as a postnatal event, extending from approximately 2 days before birth to 2 weeks after birth, whereas in humans, it begins during the third trimester of pregnancy and lasts until a few years after birth. 1 Also known as a “brain growth spurt,” synaptogenesis involves cellular proliferation, differenti- ation, and neuronal migration to target zones where formation of synapses result in the establishment of functional neuronal circuits. Neurotransmitters (includ- ing -aminobutyric acid and glutamate) and their recep- tors exert fundamental roles in neuronal migration, 2 den- dritic filopodia stabilization, and synapse development and stabilization, 3 although their role in morphologic neurodevelopment is not definitively known. 4 General anesthetics modulate specific ligand-gated ion channels, principally -aminobutyric acid type A recep- tor and the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor, thereby altering synaptic function. 5 The possibility that these synaptic actions could result in long-term consequences for the developing brain was pursued by the same group of investigators that had originally identified the deleterious effects of alcohol during neurodevelopment. 6 Therefore, postnatal expo- sure to anesthetics, which are similar to alcohol in their behavioral and molecular effects, were demonstrated to exhibit comparable morphologic and functional impair- ments. 7–9 Published evidence has shown that NMDA receptor antagonists in neonatal rats produced specific patterns of degeneration on neurons (in contrast to ame- liorative actions on the glia) 10 ; on electron microscopy, the degeneration that was noted was identical to apo- ptotic cell death. 11 Xenon has been used in clinical anesthetic practice for more than 50 yr 12 ; we reported that xenon was a non- competitive NMDA receptor antagonist. 13 Because of the strong correlation between activation of NMDA re- ceptors and neuronal injury, we surmised that xenon may act as a neuroprotectant, which we subsequently demonstrated in both in vitro and in vivo models of acute neuronal injury 14 –19 and in a preconditioning set- ting where xenon attenuates brain damage induced by hypoxic–ischemic injury in neonates. 20 Exposure to xe- non induces phosphorylation of cyclic AMP response element– binding protein (pCREB), which recruits CREB- binding protein to induce transcription of several pro- survival genes, including brain-derived neurotrophic fac- tor and the prosurvival protein Bcl-2, whose expression is increased after xenon exposure. 20 –22 In contrast, un- like xenon, nitrous oxide, another anesthetic gas that antagonizes the NMDA receptor subtype, inhibits pro- tein kinase C activity 23 and does not increase either the * Lecturer, † House Officer, ‡ Medical Student, § Senior Research Technician,  Ph.D. Student, ** Sir Ivan Magill Professor, Department of Anaesthetics, Pain Medicine and Intensive Care, # Professor in Biophysics Section, The Blackett Laboratory, and Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London. Received from the Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, London, United Kingdom. Submitted for publi- cation November 29, 2006. Accepted for publication December 6, 2006. Sup- ported by the Westminster Medical School Research Trust, London, United Kingdom, and the Medical Research Council, London, United Kingdom. Profes- sors Maze and Franks are paid consultants for Air Products, Allentown, Pennsyl- vania, a company that is interested in developing clinical applications for medical gases, including xenon. In addition, Air Products has funded, and continues to fund, work in the authors’ laboratories that addresses the actions and uses of xenon as an anesthetic and neuroprotectant. Address correspondence to Dr. Ma: Department of Anaesthetics, Intensive Care and Pain Medicine, Imperial College London, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, United Kingdom. d.ma@imperial.ac.uk. Individual article reprints may be purchased through the Journal Web site, www.anesthesiology.org. Anesthesiology, V 106, No 4, Apr 2007 746