Accepted Article Key words: C. Hirsch France; Moelle épinière Pitié-Salpêtrière, Hôpital (ICM), Boulevard F-75013, Paris, l’hôpital, de This by is protectedarticle reserved. Allrights copyright. doi: 10.1111/jnc.14041 lead to differences between thisversion the Versionand of Record.Please thiscite article as pagination which andproofreading process, may typesetting, been through copyediting, the has not but peerreviewundergonefull andbeen publication has This accepted for article 1 Lavaur Jérémie DopamineMidbrain Neurons Trophic to and Provides Xenon ProtectionStimulation Gas The Noble Article :Original type Article DR. PATRICK PIERRE MICHELID :0000-0001-5607-3119) (Orcid

l’hôpital, 75013 Paris, France. E-mail: [email protected] Corresponding author: en Josas,France. Running title: acid; ROS:reactive oxygenspecies; TH: Tyrosine hydroxylase. disease; Parkinson PD: saline; PDC:L- phosphate-buffered PBS: Dulbecco’s N-methyl-D-aspartate; NMDA: serum; horse HS: factor; neurotrophic DIV:day rhodamine-123; Abbreviations used: Xenon. Sorbonne Universités, UPMC In Sorbonne Universités, 06, Paris Univ 2 Air Liquide International Santé Medical SaclayR&D Paris, Research Center, Jouy- 1 , and PatrickP. Michel Astroglial cells; Astroglial

1 Rescue ofdopamine neuronsby xenon , Déborah LeNogue , Ara-C: cytosine Ara-C: Patrick P. Michel, ICM, Hôpital Pitié-Salpêtrière, 47, bd de bd 47, Pitié-Salpêtrière, ICM, Hôpital Michel, P. Patrick in vitro

Dopamine neurons; Glutamate; 1

; FCS: fetal calf serum; GDNF: Glial cell line-derived cell GDNF:Glial serum; calf FCS: fetal ; 1 , Marc Lemaire β -D arabinoside; DA:-D dopamine;DHR-123: arabinoside; dihydro- serm, CNRS, Institut du serm, Institut CNRS, etdelaCerveau 2 , Jan Pype Jan , trans

-Pyrrolidine-2,4-dicarboxylic -Pyrrolidine-2,4-dicarboxylic Neurodegeneration; NMDA; 2 , Géraldine Farjot Géraldine , 2 , Etienne Etienne , Accepted Article This by is protectedarticle reserved. Allrights copyright. gases". or "inert gases noble of family belongsthe to gasthat monatomic isa Xenon Introduction suggests that xenonThis mighthave some therapeutic value for disorder. this xenon can provide protection and trophic support to another noblegas notreproducexenon argon e could that Note NMDA receptors. appearedto memantine andxenonitself work by antagonizing receptor mimicked andimproved antagonist (NMDA)glutamate by theN-methyl-D-aspartate paradigm.xenon The were DAneurons ofalso effectseffects trophic in this for exerted presumably cells, mediated byglial proliferating neurons by xenonappeared indirect as it occu of DADAspontaneous,However, protection inthe the xenon. situationwas cell death where by setting enhanced DA neuroprotection Trolox alsopartially and inthis neurons rescued analog E vitamin The direct. DAwas xenon on excitotoxicity,sustained of neurons effect the as toso increase(PDC) ambientglutamate levels2,4-dicarboxylic acid slow generate and and effects onthese indirect we established that xenon waspartially protective DAfor neuronsthrough direct either or (DA) dopamine of midbrain Parkins neurons in inexperime neuroprotectiveretained its potential whether xenon to determine study, wished we Inthepresent braininjury. hypoxic-ischemic biological effects. In particular, xenonisast Despite its low chemical reactivity, the noble possesses gas xenon spectrum a remarkableof Abstract other molecules under biologic conditions(Spaggiari et This al, 2013). lackof reactivity the preventspresence of valencefrom that results filled inertness bondsa shell with covalent neurons. So, when DA neurons were exposed toL- exposed were whenDAneurons So, neurons. rong neuroprotectant in preclinical models ofmodels preclinical in rong neuroprotectant on's disease (PD). Using rat midbrain cultures, cultures, midbrain Usingrat (PD). diseaseon's rred through rred repressionthe of a mechanism astrocytes and their precursor cells. Xenon astrocytes cells. precursor and their ntal ntal settings modelthethat progressive loss ffects. Overall,ffects. present data indicate that DA neurons that are vulnerable inPD. DAneurons vulnerable that are trans -Pyrrolidine-

Xenon’s

is Accepted Article This by is protectedarticle reserved. Allrights copyright. relative somewhat 2015) 2005; 2010; Bankset al, al, brain injury (Maet ischemia,models brain in preclinical focalandglobal spinal of cordischemia and traumatic that, has xenon reported confaddition to been to Banks In 2010). 2003; etal, al, potentanesthetic (Sanders analgesic et with properties xenon anapprovedproperties ofis inhalation xenon. Forinstance, interesting biological forces (Liu etal, probably 2010; Sauguet et Suchinteractions 2016). are al, the basisof the of dopamine (DA) neurons in this disorder. More specifically, when the cultures cultures were when specifically, the More (DA) disorder. neuronsinthis of dopamine used we model midbrain settingsaim, the progressivethat two cultures loss experimental and neurodege mimic conditions that experimental 2016a,b). cholinergicforebrain neurons, aneuronal population found thatxenon Interestingly, 2016a). we et al, (Lavaur disease of Alzheimer’s to the pathogenesis relevant settingsare that in culture rescue that time first showed the we for particular, neuroprotection situations wherein neuronal the suggestednoble gas has mayalso provide it beenalso neurodegenerative that conditions, (NMDA)glutamate receptors (Liu et 2010; al, Banks al,et 2010). through indicatestudies that xenon N-methyl-D-aspartate isneuroprotective of antagonism two- 2010) orof (Bantel et al, been proposed that xenon may byoperate activation of potassiumATP-sensitive channels et3575% (Ma2008; between 2005;DavidYang2012). etal,are comprised al, etal, to These observationsThese led us toexplorethe neuroprotective xenoninpotential of While beneficial effects of xenon have been largely reported under acute under beenlargely xenonhave reported of effects beneficial While . Neuroprotective Neuroprotective effects have been generally

as xenon can interact with biological components through with interactas xenoncan non-covalent pore potassium channels (Gruss et al, 2004). Yet, a majority of majority of 2004).al, a channels Yet, (Grusset pore potassium a sustained exposure to xenonsustained provides exposureto a neuronal also worked as a trophic towards factor basal neration in Parkinson disease (PD). To this To (PD). Parkinsondisease in neration Yamamoto et al, 2010; Campos-Pires et Campos-Pireset et al,al, 2010; Yamamoto er protection againstprotection acuteer damage neuronal demise is more slowly progressing. In is demise more slowly progressing. observed in a range of concentrations that of range concentrations in a observed at disorder inthis risk (Lavaur etal,

It has Accepted Article This by is protectedarticle reserved. Allrights copyright. (AF-212-NA AbCyswas Eurobio from (Courta (L'IsleAldrich d'Abeau Glial cell neurotrophic factor line–derived Chesnes, France). (GDNF) France) Sigma- andTrolox obtained Tocris from (Lille, PDCmemantine from were and reagents Pharmacological and Material Methods antagonism. receptor NMDA antagonist memantine receptor seemedand xenon itself to operate by NMDA neurons in paradigm.this The protective trophic and ofwere xenoneffects improved by the cells. Note that in addition tobeing xenon neuroprotective, alsoDA trophically stimulated prot be to xenonappeared involving astrocytes, wheresituation DA cell wasdeath spontaneous and caused by aglial-dependent mechanism Ina culture xenonreplaced atmosphere. was thecell partially nitrogen preventedin if neurons DA theensuingthat sustained,to generate low-level lossof wefound excitotoxicity, (PDC) acid L-trans-Pyrrolidine-2,4-dicarboxylic glutamate synthetic analog the of to exposed committeeon animal experiments CharlesDarwin n°5. procedures were Experimental (2010/63/EU). Directives Union Council European and the 85-23, 1996) no. revised Publication of of Health Institutes the National Laboratory (NIH Guide Care the and Use Animals of for Animals werehoused, handled taken and ofcare inwith accordance recommendations of the Cell cultures protocols France). ;

RRID:AB_2111398) was purchased from R&D Systems Europe (Lille, R&DSystems from Europe RRID:AB_2111398) was purchased

boeuf, France). The goat anti-human/rat neutralizing GDNF GDNF neutralizing goat anti-human/rat boeuf,France). The authorized under number 0037 by ethical authorized 0037 the undernumber ective through a repressive effect on throughective these a effect repressive Accepted Article 2% to ensure optimal preservation of the cultures (Wu et al, 2009). al, preservation et the cultures of (Wu optimal 2% toensure al, 2015). Dissociated cells in suspension were then seeded at a density of 1.2-1.5 x 10 Dissociated density then 1.2-1.5 2015). cellsin suspensionwere al, of seededat a et previously 2011;Guerreiro described al. mechanically protocols et according (Toulorge to collectedAldrich) Leibovitz culturemedium andtissue in (Sigma L15 pieces dissociated a N (Gibco, Saint Aubin, France) supplemented with a B27 cocktail without antioxidants (Gibco), level excitotoxic stress, we seeded maintained and midbrain in cultures Neurobasal medium 2016). etal,8.3pH (Sepulveda-Diaz buffer, in borate diluted polyethylenimine coated with 1mg/ml This by is protectedarticle reserved. Allrights copyright. bypentobarbital and sacrificed dislocationcervical (Janvier LABS, Le StGenest Isle, France) that had been deeply anesthesized with sodium female rats Wistar from embryos using 15.5 were day gestational prepared cultures Midbrain Midbrain cultures i.e., between 0-7 DIV. From 7-14 DIV, the conc the 0-7 between From7-14DIV, i.e., DIV. phaseofculture, the initial duringthe Les France) LLC; Ulis, Eurobio, Biowest (FCS, serum seessential contained10%horse medium also minimum modified The streptomycin. penicillin and IU/ml 100 and 2mM, glutamine 2mM supplemented essential (Gibco) medium minimum Fo period. two week a over and progressively used of death inwhichthe neurons occurs DAalso culture We conditions, spontaneously 16 DIV, whenexcept indicated in the text. at terminated 12DIV and at initiated PDC with were Treatments protocol. the experimental conditions, the survivedcultures without theneed medium for culture changeend of until the cm To study the protective potential of xenon for DA neurons in a situation ofa situation low- sustained, neurons in DAxenon of potential for protective studythe To 2 ontoNunc48 plates (Therm well multidish 2 mix (Gibco), 2mM glutamine Underthese 2mMand 100IU/mlpenicillin/streptomycin. (Gibco), glutamine mix rum (HS, Sigma-Aldrich) and 10% fetal calf and 10%fetal Sigma-Aldrich) (HS, rum oFisher Scientific, Roskilde, Denmark)oFisher Scientific, pre- entrations ofHSand entrations r that, our cultures were maintained were in our cultures r that, with 1g/L glucose, 1mM sodium pyruvate, pyruvate, 1mMsodium glucose, 1g/L with . Once dissected, ventral midbrains midbrains were dissected, ventral Once

The exposure gaseousThe to FCS were reduced to reduced FCS were 5 cells/ Accepted Article gasO atmospheres (20% pre-defined supplythe Plexiglas of chambers. the set-uptechnical used incubation The for hermetically and sealed or were astrocyte midbrain placedundercultures humidified ofapplication After pharmacological treatments, multi-dish 48-well plates containing Cellculture exposure togases weretreatments for the applied 3DIV. next gaseous Then, and plating. pharmacological 1-2 growthafter after 50% days of confluency HS to andFCS Theplatingonly. de 2%, reduced ma48 wells platedonto PEIand cells were coated Sub-cultured studies. functional for subcultures scrapping produce through mechanical to days, of growth, the cells were recovered in 7-8 After FCS. HSand 10% essential 10% mediumcontaining minimum using modified indicatedconventional cellperiods incubator for time culture inthe manuscript. 37°CThe incubation maintained 2016a). were (Lavaurin a chambers at detail al, et This by is protectedarticle reserved. Allrights copyright. cm 75 uncoated onto weresuspension plated in cells Briefly, after dissection and trituration ofbr th obtained from were culture Astroglial cells in cultures. Astrocyte 14 DIV, whenexcept specified in the text. stage where neurodegeneration is ongoing.already The weregenerallycultures processed at conventionalpharmacologicalatmospheres a 7DIV, and/or wereinitiated treatments at i.e., 2 , 5%CO 2 and 75% of previouslyof has been gas)the test in and75% described ain tissue pieces in L15 Leibovitz the medium, Dulbecco’s saline (PBS) phosphate-buffered e cortices ofe cortices Wistar day-old 15.5 embryos. rat intained intheintained samemedium as before with nsity waschosen so as reach to less than 2 Nunc culture flasks andmaintained Accepted Article relative to relative Bridgewater, NJ). Results were in expressed fractional change in fluorescence intensity Corp., (Hamamatsu, software Imaging HCimage the and camera digital ORCA-ER an equipped France)with Champigny-sur-Marne, (Nikon, microscope Nikon TE2000inverted chosen were acqui fields 6randomly of images For is24hours the that eachinitiationPDC, of cultureafter fluorescenttreatment. condition, wereMeasurements made atastage where ROSis optimal exposed incultures production to beforedescribed Waltham, USA) aprocedure(Rousseauaccording MA, to 2013).et al, This by is protectedarticle reserved. Allrights copyright. permeable probe dihydro-rhodamine-123 (DHR-123) Intracellular reactive (ROS) oxygen species Quantification of reactive oxygen species quantitative assessment. fluorescence microplate reader Spectramax Devices,M4 Sunnyvale, CA)for (Molecular USA). The assay performed was according manufacturer’s tothe instructions using a acid/glutamate oxidase assay kit (#A12221) from glutamic red Amplex the weused medium, culture the ofin glutamate detection Forthe of Quantification glutamateextracellular levels cultures. presentin midbrain DA neurons to few the neuronalcells population whole of the from because ofthe technical by constraints imposed 1997-2016)was (Rasband, used quantification for Theopensource 2016). al., ImageJ al, 2011; et software Sepulveda-Diaz et probes (Toulorge

baseline in control cultures (F/F cultures incontrol baseline

0 ) as described previously for other fluorescent previously other described for ) as red with a 20X fluorescence objective 20Xfluorescence usinga with a red our model system, we ourmodel extrapolated ROSdata were assessed membrane cell were using the ThermoFisher ThermoFisher (Waltham,Scientific MA, of the fluorescent of the Note fluorescent signals. that (# D23806; ThermoFischer D23806; ThermoFischer Scientific, Accepted Article previously (Mourlevat et al., 2003). Briefly, the cultures were exposed[methyl the cultureswere Briefly, to thymidine 2003). previously al., et (Mourlevat tritiated-thymidine of incorporation the using midbrai inmixed was proliferation assessed Cell ofthymidine Incorporation tritiated Courtaboeuf, France), as previously imagingand detection conventional fluorescence respectively. imaging detection, fluorescence anAlexa secondaryfluor-555 nearinfrared Ne)and Lincoln, for antibody used asecondary to detectDAneurons. We US 1/1000 (T-9237-13; diluted (TH) antibody hydroxylase apolyclonal 4°Cstepanti-tyrosine before with incubation 24-72hours an at for 12 min using4% formaldehyde for Cultures fixed byimmunofluorescence detection Protein spectrometry. liquid scintillation PBS, thecells were recovered of the inwater for quantification radioactive signaldistilled by MEMmodified medium serum supplemenwithout This by is protectedarticle reserved. Allrights copyright. 3 to [ accumulate these neurons The functional integrity andsynaptic function of DA neurons were evaluated by theability of Measurementof DA uptake H] (1µl/ culture well, 70-90Ci/mmol, Perkin Elmer, Courtaboeuf, France) for 2 hours using 2hours for France) Courtaboeuf, Elmer, Perkin 70-90Ci/mmol, culture well, (1µl/ H]

3 H]-DA by active transport (50 nM; 40 Ci/mmol; PerkinElmer, PerkinElmer, transport(50 nM;40Ci/mmol;H]-DA byactive described (Rousseau et 2013).al, IRDye Biosciences,(LI-COR 680RD antibody Biologicals, Salem, MA;RRID:AB_2650433) Salem, Biologicals, as a marker of DNA synthesis describedas n cultures and in astrocyte andin cultures enriched n cultures in PBS, were then washed twice withPBS twice washed were then in PBS, tation. Aftertation. tworapid washes with cold

Accepted Article microscope, This by is protectedarticle reserved. Allrights copyright. also used digitized images of TH For over we thesurface well.area of each performed purposes, >60% of illustrative culture analysiswas With both methods, data softwareHCStudio (Thermoscientific). with the analyses. Post-hoc performed image analysis morphometric both cellcounting wasthen and automated fluorescence microscope (ThermoScientific, Courtaboeuf, France), thusallowing with compositeimagescell death,an XTi generated higher were Arrayscan resolution paradigm spontaneous DAof Inthe 2012). Icy etal, (deChaumont sourcethe open software semi-automatic quant 2011)and al, et (Toulorge (LI-COR imagingNe) Lincoln, Biosciences, system using infrared Odyssey LI-COR a onepiecewasof in surface scanned PDC well bottom areaeach culture In the the paradigm, Image acquisitionandcellquantification analysison ranks followedbyStudent-Newman-Keuls the spontaneouscell DAwere data deathmodel, analyzedtheKruskal-Wallis with oneway Student-Newman-Keuls followed the we data PDCsets of Inthe model, experiments. Inc,data San Jose,from USA).at leastwas pointSoftware derived 3independent Each CA, Data expressed as means ±S.E.M were analyzed using the SigmaPlot 12.5 software (Systat Statistical analyses (Hamamatsu). software HCI the with

equipped withequipped anORCA-ER digital camera (Hamamatsu Photonics), operated

+ neurons that taken with were a Nikon TE-2000 Eclipse post hoc

ification of DA cell numbers wasmade of DAcell withification re assessed by one-way ofvariance analysis test for all pairwise comparisons. In the Inthe pairwisecomparisons. all for test post hoc test. Accepted Article failed to provide protection to TH protectionto to provide failed another noblegas having poten neuroprotective 1a). rescue limited (Figure DAmore neurons, to efficacy its still but neuroprotective was xenon (>80%), more severe loss was neuronal treatment conditions where i.e., PDC, with300 4 incubation µM days of After (Figure 1b). wePDC, observed that the of loss aboveTH At (Figure 100 1a). µM to xenonwas 81%( This by is protectedarticle reserved. Allrights copyright. TH neurons (i.e., DA survival 2008). et al, The sustained of excitotoxicprocessin PD(Wallace mayoccuras it andslow a to generate PDCanalog synthetic glutamate 4days next to the the exposed for of cultures that hadMidbrain initially matured degeneration PDC-induced undergoing DA to neurons partial rescue Xenon provides Results for 1 day to 100µM PDC under a standard data Our gas a atmosphere nitrogen. under PDC containing 75% 100µM to 1day for neurotransmitt of levels the extracellular estimate wewished to 2016a), (Waagepetersen al, Lavaur 2001; Gouixetal.release et 2009; etal, BecausePDCglutamate uptake operates of its and of throughthe blockade stimulation Xenon does notinterfere with the presynaptic effect of PDC aimed to the characterizefurther neuroprotective effects ofinxenonPDCparadigm. the next We 1d. Figure in given is neurons DAfor xenon of protective effects the of illustration with 100 with treatment 1 and4 Afterdays of comprising an 75%xenon. was substituted with atmosphere largelyneurons whenthe stan protected were DA PDC-treated Interestingly, 45%to73%at1and respectivelyfrom 1b). 4 DIV, (Figure μ M PDC, the survival rate of TH of rate survival the M PDC, + cells) was significantly affected at 100µM of PDC of and at concentrationscells) wassignificantly affected at 100µM vs 55% in N in 55% 2 ; P <0.001) <0.001) and 64%( + neurons exposed to 100 µM PDC (Figure 1c). An PDC1c). (Figure 100µM neurons to exposed + neurons in cultures that were exposedchronically that were in cultures neurons in vitro dard gas atmosphere containing 75% nitrogen nitrogen containingdard 75% gas atmosphere The replacement of nitrogen argon, by nitrogen replacement of 75% The tial (Coburn et al, 2012; Zhao et al, 2016), 2016), al, 2012; Zhaoet al, (Coburn tialet er insets cultures that of exposeder been had for 12 days in serum-free conditions were serum-free conditions in 12 days for vs 27% inN 2 ; P + <0.001), respectively neurons progressed neurons Accepted Article atmosphere atmosphere containing 75% ofxenon ( an to cultures elevated in exposed remained neurotransmitter The (Figurepoint 2a). this time This by is protectedarticle reserved. Allrights copyright. ( theconcentrationshow ofextracellular that glutamate wasincreased thantwo-fold bymore (1-30µM) improved the survival of DA neurons DA survival of the improved (1-30µM) used memantine alone, When withmemantine. antagonism receptor be improved byNMDA couldprovided by xenon the rescue determine whether to wewished Therefore, PDC. 100µM As 75%xenonprovided only pa shownbefore, action of the protective xenon DA reinforces for neurons. PDC-treated Memantine 2b). (Figure NMDAreceptors ( highlymemantinewas Asexpected, death2b). DAcell against NMDA-induced effective NMDAagonist used receptors of instead wasDA of neurons PDC tokill ( the preferential Interestingly, xenonremainedpartiallywhenNMDA protective (75 µM), Xenon neurons NMDA receptor antagonism th for accounts presynaptic mechanismof ofaction PDC. thenoble mechanism throughwasprotective that tothe wasthat gas a probablydownstream neurons from degeneration ( degeneration neurons from memantine was asabout effective as 75%xenon PDC, toprotect 10 DAµM with 100µM receptors.a noncompetitive ofthese antagonist Results show thatafter 4 incubationdays of wished tocomp2012), we al, 2007; Armstrong et P P <0.001 <0.001 <0.001). This set<0.001). of data supports the view that xenon operated itself by antagonism to

having potential to operate the by antagonism of NMDA receptors (Dickinson et al,

vs control cultures under 75% N culturesunder75% control P <0.001 <0.001 P <0.001 <0.001 vs 2 control cultures under 75% N under 75% controlcultures ) after 1 day of PDC treatment to reach ~4µM, at ) PDC ~4µM, 1 dayafter treatmenttoreach of vs in a concentration-dependent manner witha manner concentration-dependent in a e rescue byxenone rescue DA of PDC-treated control control cultures under 75%N are its effect to that of memantine (10µM), that of its effect memantine to (10µM), are rtial protection to DA neuron exposedDA to protection to neuron rtial P <0.001), (Figure (Figure <0.001), 2 ) (Figure 2b). 2 ), indicating), Accepted Article protective effects for DA neurons. The blockade of NMDA receptors aprotective with ( Trolox presence 10µM of in the levels substantially reduced oxidative stress in PDµM 75% xenon( exposed to neurons PDC-treated in values to basal returned thatoxidative stress 123 revealed forown DA 3b). neurons (Figure whic of µM) concentrations higher (1-10 Trolox This by is protectedarticle reserved. Allrights copyright. underthesetreatments ofwas improved xenon Theeffect still ( conditions. neurons xenon exertedontoDA ( 75% that theaction protective stronglyreinforced µM, 0.3 and 0.1 at PDC, against protective wasnotSurprisingly, which PDC. Trolox xenon-mediatedimprove protection against all. at protection no offered µM) (10 desferioxamine whereas substantia provided (10 µM) Trolox xenon, able Fenton-type neutralize to (Thomas reactions vitamin of water-solublederivative permeable wished to protectivecompare the xenon Trolox effect of that of twocell- a to antioxidants, Oxidative stress being a component of PDC-induced neuronal death (Nafia et al we2008), treated cultures. Xenoninterferes with adeatheffector m the impact of treatments withcombined xenon improved ( further was PDC 75%xenon protective of against The effect 10-30µM. observedplateau between effect Additional Additional studies performed with the reactive oxygen species (ROS) detection dye DHR- In viewtheseIndetermine results, wished we of to whetherhad Trolox to the capacity P <0.001) <0.001) (Figure Despite3c,d). having no protective effect P <0.05) when memantine was used at 10 or 30µM. Results describing Results or 30µM. memantine wasusedat10 when <0.05) P <0.001). This<0.001). suggested asynergistic of the two action P echanism involving oxidative stress in PDC- stress oxidative echanism involving l rescue to PDC-treated DAPDC-treated neurons( lrescue to <0.001), i.e., a concentration having optimal a concentration i.e., optimal having <0.001), and memantineand are describedinFigure 2c,d. E anddesferioxamine, ferrica iron chelator h were, however, partially protective on their partially however, on h were, protective et et al, 2009). Even less if efficacious than C-treated cultures. ROS returned to basal per se per , Trolox Trolox 0.1at , P <0.001) by <0.001) P <0.05)

Accepted Article This by is protectedarticle reserved. Allrights copyright. of TH rate survival the conditions; show xenon that provided partialbut ( significant we at7 started engaged, well our treatments byalready whenneurodegeneration DAcellrescue xenoncan determining whether is occur wewereBecause 4a). in particularlyinterested (Figure 95%byDIVDIV 14 than andmore stagesdifferent of the cultures, that 7 DA abouthalf weestablished of lostat were neurons spontaneously function and progressively asa inwhichsetting DAneurons in serum-supplemented maintained degenerate culture medium used potential xenon, we the anotherexplore experimental of neuroprotective To further supplemented midbrain cultures. Xenon is also protective for DA neuron Noteofnone that the above reduced treatments ROSbelow levels controllevels. alsosuppressed ROSproducedby PDC( memantineconcentrationµM) of (10 4c). (Figure cultures cell culture atmosphere also 75% xenoninthe 14 failed tomodify in DIV glutamate levels loss inthis experimental context was probably unrelated to Theexcitotoxicity. presence of low 7DIV marginallyat and increased at 14 DIV (1.15 glutamate were concentrations extracellular show that Our results 14 cultures. DIV control slowly progressingand process, excitotoxic of wecompared the neurotransmitter in7 levels (Figure 4b). settingsame the survival in impact onDAcell significant no had containing75% argon chronically exposed Note xenon.to 75% of that exposing thecultures to an atmosphere To determine whether xenon operated, here, like in the other setting by interfering with a setting in other like with by xenon the whether here, interfering determine operated, To + neurons was 14 DIV cultures neurons increased by194% in s that spontaneouslyare dying in serum- DIV and stopped them at 14 DIV. Our results at Our them 14DIV. DIV andstopped of time. Whenof time. monitoring numbers DA at cell P <0.05) rescue to DA neurons in these DA neuronsin to rescue <0.05) vs 1.4µM), indicating DAcell that 1.4µM), P <0.001). <0.001). Accepted Article of DA uptake of DAwas uptake expressed relative toTH bywell ( 333% 14DIVcultures in DA/culture gasxenon thatneurons, atmospherea wefound increasedin of enriched the uptake This by is protectedarticle reserved. Allrights copyright. found that TH Even the if spontaneous loss DAof neurons was apparently notexcitotoxic innature, we serum-supplemented midbraincultures. Xenon and memantinecooperate to protect DA Guerreiro et et 2015).Guerreiro al, with theCoherent of survival xenon promoting effects TH for assessed their ability to accumulate [ accumulate to ability their assessed ( conditions the is ofculturesreached casethat it 652% inrate ofDAneurons asthe these survival control clearlyshow results Our DA neurons. of survival improve the further memantine wasableto treatment and combining spontaneously wetested a neurons, whether xenon dying DA rescue to provided partial memantine Because treatments with 75%xenonor10µM either to however, against PDCpartially protective was, was which (10µM) Trolox control cultures. age-matched to comparison memantine in memantine (279%) ( memantine (279%) (204% xenon on than ofthis memantineparameter ( wasgreater that only preserve to survival theof DAneurons but tostimulate also their (Figurefunction 5b). conditions 1191% of ( control values conditions these which in reached improvedDA uptake, further under75%xenon, with memantine atmosphere containingcontrol 75%nitrogen ( DAcultby wasincreased midbrain 487%in We next studiedwhether next We DAneuronsrescued Forthat weby xenonwerefunctional. vs 163%, respectively). Again, the effect of xenon appeared further enhanced by enhanced appeared xenon further of respectively).the effect 163%, Again, P <0.05 + cell cell numbers were increased by 407% ( vs P each individual treatment) (Figure 4e). <0.05). Altogether, <0.05). these results show that xenon the has ability not P <0.05 <0.05 3 H]-DA by active transport (Rousseau 2013; (Rousseau et al, H]-DA transport by active

vs + P each treatment). individual themeasure When cell numbers (figure 5b), the effect of 75% effect of the 5b), (figure cell numbers P ures treated with 10µM memantine under memantine a ures treated with10µM <0.05). Of interest, performing interest, treatment Of the <0.05). <0.05) (figure 5a). Meanwhile,5a). the (figure of uptake <0.05) tally ineffective in this setting (Figure 4d). 4d). (Figure setting inthis ineffective tally neurons arespontaneouslythat dying in P <0.05) in the presence of 10µM in <0.05) presencethe of 10µM P <0.05) under 75% nitrogen under75%nitrogen <0.05) +

Accepted Article This last set of observations suggested that GDNF, a peptide that exerts survival and trophic trophic survival exerts and peptide that GDNF,observations a setof that suggested This last involvedofGDNF isnot intheeffects xenon neurons. ( by parameter enhanced this xenon or 75% memantine performed under gaseous containing with atmospheres either 10µM 75% nitrogen GDNF antibody (AB212 NA; 10 anti- an that we found Yet, setting experimental 6a). (Figure in this 75% xenon protection as At effects. an optimal of 20 concentration ng/ml, GDNF provided thesame level of midbrainLiss, DA effects for and neurons (Kramer waspossibly 2015), xenon’s involved in density/TH Consiste memantine. presence 10µM ornotof DIV cultures midbrain under maintained control orxenon-enriched atmospheres in the This by is protectedarticle reserved. Allrights copyright. TH quantified the totaldensity of due trophic to the action gas thenoble on of neurons. Toaddressthese this wequestion, whether inducedby inDAuptake xenon was thewe increase wished to determine 1995), al, on being located sites DA preferentially uptake onspontaneously effectsXenonneurons trophic dying DA exerts antibody. memantine, alone, or in combination with xenon remained unaffectedalso by the anti-GDNF of xenon(Figur that reduce failed to peptide,

P <0.05). Figure 5d illustrates Figure theimpact<0.05). of previous treatments on the morphology of DA + neuron was neuron increased ( byunder 75%xenon 179% μ + neurites with respect neurites with of to the 14 number DA in neurons g/ml) able to neutralize the protective effect of the trophic trophic the of effect protective the neutralize to g/ml) able 262% respectively(Figureand 337%, 262% 5c) e 6a). Importantly, the rescue provided by provided rescue the Importantly, 6a). e neuritic extensions of DA neurons (Ciliax neurons et ofDA extensions neuritic nt with nt with our hypothesis, total neurite the P <0.05). Besides, treatments Besides, <0.05).

Accepted Article 6d). xenon atmospheres. remained Memantine,alone and bothnitrogen effective in equally remained detectedin these conditions. Ara-C (10µM) werealsomemantine xenon to 10µM 75% combined of 75%xenonandeffects of that proliferation out carried assays us additional represent a likely xenon-mediated for target ne cultures midbrain supplemented withserum(M effect on glial cells in itself. these( cultures for gas theDA neurons(Figure noble action also reinforced the of 6b), in anti-proliferative memantine, which of 75% strongly therescuing effect xenon improved Of interest, 10µM survival of TH of survival by ( 53% cell proliferation glial wasreduced Ara-C, 10µM presenceof This by is protectedarticle reserved. Allrights copyright. in cultures that hadbeen maintained under75% N Ara-C drug ( with antimitotic the proliferation conditions. Insupport to under present this po the repression ofadeath mediatedbymechanism glial cells, proliferate which spontaneously result could from xenon the action of the addressed neuroprotective We next possibility that mechanism. dependent of fromDA Neuroprotection by results neurons the repression xenon of a glial- significantly reduced( significantly appeared to be improvedalso by75% xenon, although,not significantly. Astrocytes or their precursor cells whichar cells precursor their Astrocytes or Consistent with our working gl hypothesis, P + neurons improved by 435% ( 435% improved by neurons <0.05) (Figure 6c). Surprisingly, memantine (Figure however, norepressive exerted <0.05) 6c). P <0.05) in midbrain cultures maintained under 75% xenon (Figure 6c). 6c). (Figure 75% underxenon cultures maintained in midbrain <0.05)

10µM), led to a betterto DAneurons survival of a led 10µM), ing astrocyte cultures. The ing astrocyte cultures. anti-proliferative uroprotection in thepresent uroprotection Thus, wesetting. ssibility, we showed that reducing glial cell glial that reducing showed we ssibility, e the most abundant types of glial cells in cells abundant glial types of most the e ourlevat et al, 2003;Rousseau etal, ourlevat 2013),et al, P totally inactive in astrocyte cultures (Figure astrocyte inactive in cultures totally <0.05) (Figure 6c). The effect of Ara-C of The effect 6c). (Figure <0.05) ial proliferation cell was slightly but 2 between 7-14 DIV (Figure 6b,c). In the Inthe between 7-14DIV(Figure 6b,c). P <0.05) and <0.05) the Accepted Article This by is protectedarticle reserved. Allrights copyright. PDC by induced stress excitotoxic low-level sustained, from neurons DA protects Xenon we used. DA onto effect indirect or direct either a antagonizing glutamateNMDA receptors. Xenon exerted protectiveits action by means of NMDA receptor antagonist memantine and the noble gas itself appeared towork by PD.these neurons in In the of cases, both xenon wasmimickedeffect and improved by the experiment two distinct neurons in DAmidbrain tobutsustainedprotection but that notargon xenon heredemonstrate We provides partial Discussion 2016a). At 100µM of PDC, a concentration killing about 75% of DA neurons over a period PDC, DA killing over aperiod of aconcentration 75%of neurons about At 100µM 2016a). operates through inhibition of glutamate transport 1994;Blitzblau More PDC et 1996). precisely, al, (Maki al, et milieu the extracellular glutamate receptors on its own midbrain PDC,cultures toastructural analog of glutamate that doesnot act anagonist as of 2013). etal, (Vaarman via D via decrease presynaptic the that thene inhibition Char 1999; etal, glutamatergic neurons(Piallat dopa reduced itself from mayresult This process 2016).al, et2008; Michel et al, 2001; Wallace al, et glutamatergic neuronsnucleus (Blandini process occurring asa consequence of an increased excitatory drive from subthalamic suggest reports Aof DA number cell aslowexcitotoxic death that resultfrom inPD may To To model a slow and sustained excitotoxic process as itmay occur wein PD, exposed .

2 receptors, thus making these neurons further vulnerable stress to excitototoxic thus neurons further making these receptors,

but does generate toxic build-up of a the in neurotransmitter neurons depending upon the experimental paradigm paradigm the experimental upon neurons depending urotransmitter directly exerts ontoDAneurons les et al., 2008). deficit al., The inDAmayalso les et minergic inhibitory control over subthalamic subthalamic over control inhibitory minergic al settings that model the progressive loss of loss progressive the model that alsettings and stimulationofrelease its (Lavauret al, Accepted Article This by is protectedarticle reserved. Allrights copyright. when noble strongly the protective remained gas possibility, Supporting receptors. this neurons exposed indicating toPDC, xenon that itself by may of operate antagonism these Memantine, an antagonist of NMDA glutamate receptors was like xenon protective for DA receptor blockade The protective ofeffect xenon for PDC-treated DAneuronsmediatedby is NMDA the present setting through a direct ontoeffect DA neurons. in culturesexposed to75% Thisworked means noblegas xenon. thatthe post-synaptically in unlikely theincrease inas glutamate extracellular by levels produced same the remained PDC a pr from possiblytreated DAneuronsmay result PDC- for xenon effect theof neuroprotective that suggested This 2003). etal., (Petzelt milieu th reduce its ability to neuroprotective through tobe reported was neuronal xenon damage, system ofhypoxia-induced In amodel of PDC action presynaptic with the interfere not does Xenon neurodegeneration. the protectiveeffect of xenonisdescribedin that time first the is this However, 2014). al. Deng et 2009; al. et 2003; Thoresen al. David et paradigms that acuteneuronal model insults previous neuroprotective of works xenon potential reporting onthe inexperimental by with was modified replacing 75%nitrogen neuronsfor DA2008). (Nafia setting deleteriouspotentially et this in al, experimental confirming view that the sustained alimitedof but elevation extracellular glutamate was of 4day, we noted that extracellular levels of glutamate were increased by two-fold, DA cell death induced by PDC was substantially reduced when the cultureatmospherecell when PDCsubstantiallyreduced induced by was death DA cell 75% xenon. This observation is in line with is Thisobservation line in xenon. 75% in ischemic conditions (Banks et al . 2010; al. (Banks et conditions inischemic a paradigm that isa paradigmPD-induced relevant that to e releaseglutamate of e extracellular inthe esynapticeffect of noble gas. the This was

Accepted Article This by is protectedarticle reserved. Allrights copyright. cont present In the 2013). al, (Brennan-Minellaet damage neuronal stress-mediated turn stimulates oxidative in which incytosolic calcium rise ofThe overstimulation NMDA resultsreceptors series ina ofdownstream events, notablya neurons. Xenon reducesimpact the ofoxidative stress-mediateddamage inPDC-treatedDA with NMDA receptors and protect DA neurons. xenon (Zhang 1995) may intheabilityXu, a keyelement of noblegasand be this to interfere shell, did not protect DA PDC. neuronsfrom Th why memantine enhanced the protective action of xenon for DA neurons. (Lipton al, 2007; Kotermanski et and Johnson, 2010),2009; Liu etal, which may explain of memantinebe that distinct xenonfrom to appears binding for activesite the any case, has which through binding site, tobe another Infunctionally 2010). al, characterized (Liuet under these means conditions. This xenonthat with NMDA interfere may receptor function glycine site, which indicates that xenon may be unable to compete efficiently glycinewith paradigm, glycine aconcentrationcontains of that issaturating for theNMDAreceptor et (Harris at the NMDA receptor modulator, 2012). NMDA al, (Armstrong et evokedby inwardcurrents hasto reduce xenonshown thecapacity have NMDAreceptor that 2010).al, Besides, studiesfunctional on lines performed cell overexpressing the subunitsof stimulations that predict putativeseveral bindi with dynamic molecular also coherent xenonis of action for a mechanism degeneration. Such NMDANMDA, aprototypical agonistfor recept Noticeably, another noble gas argon, which shares in common with xenon a filled valencewithfilled xenon a shareswhich argon, gasin common another noble Noticeably, More specifically, xenon hasbeenreported compete to glycine, with a positive allosteric al, 2013). Neurobasal medium used in the PDCthe usedin Neurobasal medium al, 2013). ng sites for xenon on NMDA receptors (Liuet onNMDA receptors ng xenon for sites ext, oxidative stress may also arise from the from also arise may oxidativeext, stress is means is that the larger atomicradius of ors, replaced PDC to trigger DA neuron replacedDA PDCto triggerors, Accepted Article implicated (Jenner,implicated 2003;Salazar et al, 2008; Guerreiro et al, 2009). particularly relevant for diseasea state such asPD stress where oxidative appears crucially efficacy xenon of against lowandsustained excitotoxic insults. These observations may be se per protective not Trolox were that low concentrationsof DHR-123that revealed with performed PDC,against wereto substantiallyable improve theprotective action of 75% Studies xenon. neurons. Unexpectedly, however, concentrations DA for protection providingpartial enhancedxenon wasof Trolox by concentrations of the effect that protective We found effect. this had to improve the Trolox capacity whether This by is protectedarticle reserved. Allrights copyright. 2015). substrate thesynthesis for majorthe cellul of neuronal glutamate a 2011), system th transport blockade by al, (Assous etal excitatoryet 2014; acidcarrier-1 Berman PDC of the amino al, 2016a). (Lavaur et properties xenon,antioxidant whichlacks like by memantine, inhibited was also overstimulation.NMDA receptor with Coherent thisview, stress oxidative by induced PDC itsto limit indirect to was capacity andsolelyROSxenon due on impact of production unlikely as to operate an antioxidant on its own (Lavaur 2016a),et al, which suggests that the possessproperties.We intrinsic antioxidant suggestingxenon may Trolox, that 10treated neurons exposed 75%xenonorto µM to probefluorogenic DHR-123 that oxidative revealed returned tobasal values stress in PDC- The of use the reduce DA demise cell when used an of optimal concentration at 10 µM. had thecapacity thefreeobserved radicalscavenger 2014) to and Muller, (Janc that Trolox Because Xenonprovided onlyprotectio partial we PDC, deathinduced by DA oxidative cell in that stresswasinvolved Confirming

had, however, a small antioxidant effect which seemed sufficient to reinforce the toreinforce seemedsufficient effect had, however,antioxidant which asmall have was however, xenon that shown previously, ar antioxidant glutathione (Aoyamaglutathione Nakaki, andar antioxidant of Trolox providing no protection at at all protection no providing Trolox of at co-transport cysteine, the rate-limiting rate-limiting co-transport cysteine, the at n DA to neurons,PDC-treated we asked Accepted Article antioxidant Trolox, which provided partial protection against PDC-induced DA cell death,against DA PDC-induced cell protection partial provided which Trolox, antioxidant wellprobably threshold. excitotoxic i.e., underthe This also why explains the µM, below 1.4 7- between (i.e., cultures midbrain applied to during increased only marginally were levels unlikely the toaccount for spontaneous loss of DA neurons.Indeed, extracellular glutamate of origin ga the theprotective noble of effect the also,here, at was NMDAantagonism that speculation receptor the leading to memantine, by of xenon was effect the protective amplified that established we Interestingly, system. promoteDAcellsurvival model same antagonists showing inthe NMDA receptor that iswhich nottotallywe unexpected work refer tothe if Wu from (2009)and colleagues This by is protectedarticle reserved. Allrights copyright. conditions. also inthe previous one wasdueto the slowly evolving the natureof insults in these two onemaythat itslack2016), assume Zhao al, et against is stronglyAs neuroprotective Argon protective spontaneouslyfor dyingneurons DA treatments can bepossibly administered tothem. whereDamier neuroprotective1999) and al, 2012; et 1997;Jellinger, etal, (Ma patients clinicalsymptoms become where inapparent point hasreached a ~50% is loss DA that cell more progressive the one. previous Besides, PD than allows modelling where it for stage a platingprogressively 2009).(Wu etal, This after interesting is paradigm is it even in that loss the conditions which in experimental usedwe potential offor DAneurons, xenonmidbrain theneuroprotective explore To further Xenon protectionprovides to spontaneously dying DA neurons Memantine like Memantine like xenon afforded partialprotec towhatweobserved paradigm, previous we inthe that xenonSimilar found waspartially the time period during which treatments treatments were during which period the time of these neuronsof occursspontaneouslythese and s. A slow excitotoxic process was, however,was, process excitotoxic s. Aslow 14 DIV) and these remained concentrations these 14DIV) and acute ischemic acute (Brücken 2013; insults etal, of protective effect in the present setting and setting inthe protective effect present of whereas argon remainedwhereas totally ineffective. tion to spontaneously dying DA neurons, Accepted Article This by is protectedarticle reserved. Allrights copyright. repressive effect of the gasnoble towards astroglial Xenon cells. had a modest inhibitory possibly we that hypothesized result of the was rescue Thus, by a the xenon to DA neurons. protection some provided Ara-C, the antimitotic with proliferation cell glial asreducing here, 2003;Toulor for etal, DA (Mourlevat neurons astrocyt reportedto render of culture condition maintained the Spontaneouslyof proteins, in presencea were serum dying DAneurons cells astroglial througharepressive neurons spontaneouslyeffect on Xenon dyingDA protects culturesof midbrain treated (notshown). with xenon medium culture inthe undetectable remained GDNF observations, with these line xenon. In to significantly protective the reduce effect of with alone orin memantine combination thesame antibody Notethat effects of failed also the noble gas. the probably involved in not GDNF, providedexogenous cell rescue whichi by was resistantof xenon to 2011) a neutralizing al, DAantibody thatprevented et (Toulorge to support and trophic protection xenon provided both xenon’s effects in the present setting.GDNF to Similar Liss,what and (Kramer 2015), does Therefore, possibilit 2009). et al. the we studied thestimulate synthesisthe of G factor trophic The twoNMDA receptor antagonists ifenprod neurons DA dying spontaneously for xenonof effects protective the to notcontribute does GDNF actionxenon of spontaneously for dying we explored neurons, other possible mechanisms. remained totally ineffective inthe present paradigm. Therefore, to explicate the protective DNF astrocytes in (Toyomoto etal, 2005;Wu es and their precursor cells potentially harmful harmful cells potentially precursor their es and y that GDNF could operate as a mediator of amediator as operate y GDNF could that ge et 2010).Thisal, was probably case,the il and memantine have been reportedto ndicates that this trophic factor was most was factor trophic ndicates thatthis DA neurons. Yet, the protective effect theprotective Yet, DA neurons. Accepted Article This by is protectedarticle reserved. Allrights copyright. (Michel et al, 1997; Mourlevat et al., 2003). This means that the small ofeffects xenon-based also betterdi but survival better not onlyin ofthe repressive the action reporting that Th memantine. not with or associated by xenon, we also setting, found In this thatDA neurons and trophically were functionally stimulated 2017). Liddelowet al, 2013; (Rousseau etal, demise permissive for DAcell were tobe reported cells astroglial by proliferating secreted 2013;Liddelowal, et Notably,al, soluble2017). including variety degenerativeactors of death neuronal of ina conditions, PD (Rousseauet perspectivefindings other in with be placed events are andneuronal glial how demonstrated to be remains it Jimenez-Blascoif Even2015). etal, 2007; Kirchhoff, (Verkhratsky and precursor cells, presumably through blockade to the capacity the of two treatments toreduce proliferationthe of ortheirastrocytes t alone orxenon protection provided by xenon, of that the signifies inboth level This byand astrocytesmidbrain memantine cultures. was, enhanced however, cells ofglial xenon anti-proliferative effect towards The be proven. to totallyexcluded butremains isnot proliferation cell on withnoimpact function astrocyte cellglioma proliferation (Ramaswamy et al, 2014). Apossible of effect memantine on somehow surprising as MK-801, NMDA another receptor blocker was reported to reduce system exerted, however, no anti-proliferative effect which Memantinemodel1999). more potent thisneuronsprotecting DA in was xenonin than showing that xenon can slightly delay cell cycle pronounced inpure cultures, is astrocytic which slightly was more this and cultures in midbrain effect glial of cells on proliferation effect the antimitotic Ara-C towards astroglial cells, resulted Ara-C cells, antimitotic towards astroglial fferentiation of DA neurons in midbrain cultures of DAneuronsinmidbrain fferentiation of functional NMDA receptors on these cells these NMDA on receptors functional of showing that reactive astrocytes may be key key be may astrocytes reactive that showing ogether with memantine was with possibly related ogether is observation is reminiscent of earlier studies earlier of is reminiscent is observation mechanistically presentdata interrelated, can progression in astrocytic cells (Petzelt et al., (Petzelt astrocytic et cells in progression toxins or extracellular orextracellular matrix toxins components also consistent with a previous report report previous with a consistent also per se inmidbrain whichcultures, is Accepted Article editor ofJNC. This by is protectedarticle reserved. Allrights copyright. deNationale la Recherche de laet Technologie (ANRT employees. DLN was supportedbyCIFRE docto a assistance regarding automated fluorescence mi Mo atICM. culture facility, the CELIScell for inNeurosciences.Biotherapies equipment from work This and services benefited from andANR-11-INBS-0011-NeurATRIS: 10-IAIHU-06 to these results also received th supportfrom leadingThis studyLiquide by Theresearch SantéInternational. grant wasfunded Air from a disclosure interest of conflict and Acknowledgments tempting to speculate about the therapeutic value of xenon in pathology.the indirect mechanisms that implicate probably astr through also DA and neurons onto effect direct through a potentialtooperate hadthe Xenon protection and trophicto exert or restorative effe mechanisms xenon ofactionthe for in present modelsystem. effects elicited by such treatments, even if we cannot totally otherexclude possible on proliferativebe astrocytes maytreatments applications for the use of xenon disease. Parkinson's useof for as therapeutic the agent xenon for applications support with gas delivery withgassupport systems. projectMatthieu for Milet François management Aude and Chalopin and technicalGilles for We Healthcare. the wish also tomention contribution staff otherAir members, of Liquide Altogether, present data demonstrate that the noble gas xenon has the ability to provide abilitythe to has xenon gasnoble the present that datademonstrate Altogether,

Potential conflicts of interest: Air Liquide has filed patent has filed AirLiquide of interest: conflicts Potential e programs “Investissements d’Avenir” ANR- re specifically, we thank David re specifically, Akbarfor at the origin of both protective and trophic trophic and protective both of origin the at cts DAfor neurons that are vulnerable in PD. croscopy. ML, JP and GF are Air Liquide Air GFare ML, JPand croscopy. ocytes. On the basis of these results, it is ral fellowship co-funded by Association byAssociation co-funded fellowship ral Translational Research Infrastructure Translational , 2014/0589) andAirLiquide

E. Hirsch an is Accepted Article This by is protectedarticle reserved. Allrights copyright. byinhibition xenon without binding.affecting glycine N-methyl-D-aspartateglycine-binding receptor that selectively site competitive prevent Franks Dickinson N.P., R. (2012) of two Identification mutations (F758W and F758Y) in the T.J., BanksArmstrong Geldart Simillis McKitrick C.H., EdgeC.J., R., S.P., P.J., Babla C., (EAAC1). 1 Acid Amino Carrier Excitatory the Aoyama T. Nakaki K., Homeostasis: Redox in Cellular (2015)Glutathione Association with References acid transporters in the in acid transporters dysfunction by Kachidian (2014)ProgressiveL., acute excitatory P. of Parkinsonism amino Mel P., Gubellini L., Had-Aissouni M., Assous Anesthesiology receptor mediates N-methyl-D-aspartate P., R.inhibition glycineBanks (2010) Competitive Dickinson atthe of N.P., the site Franks sensitive potassium channel opener. Bantel C., Maze TrappM., S. (2010) Noble Parkinson's disease. prevents basalganglia of metabolic changes model andnigral in a degeneration rodent an ofantagonistNMDA Subthalamicinfusion (2001) Greenamyre J.T. Nappi G., Blandini F., neurons the EAAC1-/-in mouse. Edling Swanson Y., (2011)N-acetylcysteineT.M., R.A. loss prevents dopaminergic of Berman A.E., Chan W.Y., Brennan A.M., Reyes R.C., AdlerSuh B.L., KauppinenS.W., mediated neurotoxicity inrat cultures. cortical L-trans-pyrrolidine-2,4-dicarboxylateinhibitor transportevokes indirectlyreceptor NMDA S.,Robinson M. GuptaBlitzblau R., Djali S.,

112 , 614-22. Ann. Neurol. rat substantia nigra.rat substantia

Ann. Neurol. 49 Anesthesiology , 525-9. xenon hypoxia-ischemia. against neuroprotection Neurobiol. Dis. Neurobiol.

Eur. J. Neurosci.Eur. J. 69 gas xenon is a novel adenosine triphosphate- Molecules on C., Nafia I., Nafia on C., Salin P., Kerkerian-Le-Goff B., (1996)Rosenberg Theglutamate B., P.A. , 509-20.

112 Anesthesiology , 623-30.

20 65 , 8742-58. , , 69-81.

8 , 1840-52.

117 , 38-47. , Accepted Article Levey A.I. (1995) The dopamine transporter: immunochemical characterization and characterization transporter: immunochemical dopamine The (1995) Levey A.I. H.B., Niznik Ince Hersch Z.B., E., S.M., Pristupa L.L., Heilman C., Demchyshyn Ciliax B.J., 424-6. PD? in thecourseof applied neuroprotective early if stimulation CharlesGill P.D., DavisC.E., Konrad T.L., Benabid (2008)P.E., A.L. Is deep brain traumatic brain injury. outcome neurologic and reducessecondary in an invivo following trauma of injury model V., Pankajakshan P., Lecomte T., Olivo-MarinLagache J.C.MontagnerPankajakshan P.,Dufour Lecomte A., Y., T., Le V., de ChaumontF.,Dallongeville S.,S., ChenouardN.,Provoost Pop HervéT., Meas-Yedid N., subjectedopportunity inrats to transient cerebral ischemia. Neuroprotectiv J.H. (2008) Abraini M., Lemaire David H.N., Haelewyn B., Rouillon C., Lecoq M., Chazalviel L., ApiouG., Risso J.J., D(28K) immunohistochemistry. Nigrosomes on organization calbindin I. compartmental brain. and nigral a based matrix, the Hirsch Damier E.C.,P., A.M.Agid Graybiel Y., (1999)Thesubstantia nigra the of human 'lazy'the noblegas properties.with organoprotective (2012)Argon: Magalon Rossaint G., Rex Fries M., R. R.D., Ma D., S., Sanders Coburn M., localization inbrain. This by is protectedarticle reserved. Allrights copyright. Franks N.P., R Engelhard Dickinson Thal Werner C., T., K., S.C., Campos-Pires Armstrong R., Sebastiani Gruss S.P., M., Radyushkin A., LuhK., Hirnet C., recovery after cardiac arrestinrats. (2013)Argonreduces FriesM. G., Brücken A., Cizen A., FeraMeinhardt C., A.,Weis J., Nolte K., Rossaint R.,Pufe T., Marx Dis. Death kinase couples NMDA receptors tosuperoxide Brennan-Minnella A.M., El-Benna Shen Y.,

4 , e580. J. Neurosci. Crit. Care MedCrit. Care Brain Brain

15 neurohistopathological damage andpreserves neurohistopathological functional damage Br. J. Anaesth. J. Br. , 1714-23. 1714-23. , . 122 43 , 149–158., , 1421-36. J., J., Phosphoinositide 3- Swanson(2013) R.A. release in excitotoxic neuronal death. neuronal in release excitotoxic e effects of xenon: a therapeutic xenon: of windowof a e effects

110 Eur JAnaesthesiol. Eur , Suppl, 1:i106-12. FASEB J. FASEB Nat. Clin.Nat. Pract. Neurol. . (2015)Xenonimproves

22 , 1275-86. ,

29 , 549-51. , Cell

4 , Accepted Article (2014) Neuroprotective gases-fantasy or reality for clinical use? use? gases-fantasy forclinical orreality (2014) Neuroprotective Deng J., Lei C., Chen Y.,Fang Z., Yang Zhang Q., H., Cai M., Shi L., Dong Xiong H., L. Methods This by is protectedarticle reserved. Allrights copyright. electrophysiology. receptor by xenon theanesthetics isoflurane:and evidence from molecular modeling and (2007) Franks Competitive N.P. inhibition at the glycine site of the N-methyl-D-aspartate Dickinson R., Peterson Banks P.,B.K., Simillis C., Martin J.C., Valenzuela C.A., Maze M., 45. pl informatics openbioimage an (2012) Icy: metabolism uricmetabolism acid:by potentiation low-level depolarization. (2009)Protection P.P. midbrainof dopaminer Michel E.C., Hirsch D., Alvarez-Fischer E., D., Toulorge Martin A., Ponceau S., Guerreiro cyclopropane. domain K Gruss Bushell T.J., LiebM., Bright D.P., W. activation. death through extrasynaptic cell NMDA uptakeneuronal receptor glutamate glial triggers Léveillé O., F., Gouix MelonC., Nicole Had-AissouniE., Buisson L., (2009) A. Reverse inhibition at the N-methyl-D-aspartate receptor glycine site. ismediated argon, by butnot brainby injury against xenon, Neuroprotection traumatic Dickinson (2013) Franks R. Kiru N.P., L., R., Campos-Pires Armstrong K., S.P., Harris alone or in cooperation with nicotine. sleep-modulating peptide orexin-B protects midbrain dopamine degeneration,neurons from The (2015) Michel P.P. E.C., Hirsch S., Hamadat RousseauFlorence E., C., S., Guerreiro

9 + , 690-6. 690-6. , Mol. Cell. Neurosci. channels are a novel target for the anesthetic gases xenon, nitrous oxide, and oxide, xenon, nitrous gases the anesthetic for a novel target arechannels Mol. Pharmacol. Anesthesiology

40 65

, 463-73. , 443-52. 107 Mol. Pharmacol. , 756-67. atform for extended reproducible research.atform extendedreproducible for R., MathieA., FranksN.P. (2004)Two-pore- gic neuronsby the end-productofpurine

87 , 525-32. Anesthesiology. J. Neurochem. Prog. Neurobiol.

119

109 , 1137-48. , 1118-28.

115 , 210- , Nat. Nat. Accepted Article This by is protectedarticle reserved. Allrights copyright. (2009)Mg S.E.,Kotermanski JohnsonJ.W. pathway. Astrocyte NMDA receptors' activity sustai Jimenez-Blasco D., P., GonzalezSantofimia-Castaño Almeida A.,A., (2015) Bolaños J.P. discussion S36-8. changes of concepts. spor of (2012) Neuropathology K.A. Jellinger model of Rettsyndrome. amouse in tolerance hypoxia plasticity, andimproves synaptic reinstates hyperexcitability, scavenger neuronal Troloxdampens radical free M. O.A., Müller (2014)The Janc the Alzheimer's drug memantine. Jenner P. (2003) Oxidative stress in Parkinson's disease. disease. Parkinson's Oxidativestress in (2003) P. Jenner Discov. mediated inresponse tosuneuroprotection Xenon- (2016a) Michel P.P. E.C., Hirsch Le Lemaire Nogue D., J., M., PypeLavaur J., disease. for Parkinsonimplication Kramer E.R., Liss (2015) signal GDNF-Ret B. Neuroprotective Neuroprotective and neurorestorative potential of xenon. Lavaur J., Stevens B., Barres B.A. (2017) Neurotoxic re Stevens Barres (2017) Neurotoxic B.A. B., B.A., Panicker N., Kumar M., BuckwalterM.S., Rowitch D.H., Dawson V.L.,Dawson T.M., Chung MünchBennett Pete M.L., W.S., A.E., L.,Schirmer Bohlen C.J., F.C., Bennett L.E., Clarke K.A.,Guttenplan Liddelow S.A., microglia.

2 , 16018. Cell Death Differ. Nature.

Lemaire M., Pype J., Le Nogue D., Hirsch E.C., Michel P.P. Michel (2016b) HirschE.C., Lemaire PypeLe Nogue D., M., J., 2017 Mov. Disord. 541 Front. Cell.Front. Neurosci.

, 481-487. , 22 , 1877-89. 1877-89. , J. Neurosci.

FEBS Lett. FEBS 27 , 8-30. 2+ stained, low-level excitotoxic stress.

imparts NMDA receptor subtype selectivity to subtype selectivity NMDAreceptor imparts 589 29 ns survival neuronal through aCdk5-Nrf2

ing in midbrain dopaminergic neuronsandits dopaminergic ing inmidbrain 8 rson T.C., Wilton D.K., Frouin A., Napier Napier D.K., FrouinA., Wilton T.C., rson , 2774-9. , 56. 56. , active active by induced astrocytes activatedare , 3760-72. adic Parkinson's disease: Parkinson's evaluation andadic Cell Death Dis. Dis. Death Cell Ann Neurol.Ann

53 7 , e2182. , Suppl3:S26-36; , Cell Death Accepted Article This by is protectedarticle reserved. Allrights copyright. Disease. Parkinson in HirschMichel E.C., P.P., Hunot S. (2016) U 3414-23. disector counts. inParkinson's nigra features using substantia clinical disease and neuromorphometry inthe Ma S.Y., Röyttä M., Rinne J.O., Collan Y., U.K. Rinne (1997) Correlation between neonatalasphyxia. neuroprotection from A.D.,Franks N.P.,M. Xenon provide Maze to hypothermia (2005) and combine Edwards H., Mehmet R.D., Sanders R.M., Battson M., Arshad A., Chow M., Hossain D., Ma receptors from MD simulations. Tang of(2010) Mechanistic NMDA XuY., intoxenoninhibition insights Liu P. L.T., NMDAreceptorby block S-nitrosylation. and memantine (2007)Pathologically-activated therapeuticsLipton of neuroprotection: mechanism for S.A. repression of presumptive astrocytes. neuronal death by cyclic AMP neuron inmixed Troadec S.,Mourlevat RubergJ.D., Mich M., disease. Parkinson for tosurvive: in implication stimulation order for neurons dopamineneeds of (2013)Specific S., HirschE.C. Guerreiro Toulorge D., P.P., Michel mechanismin hippocampalneurons. cultured excitato depressespyrrolidine-2,4-dicarboxylate R., RobinsonM.B.,Maki L-trans- glutamate M.A. Dichter (1994)uptake inhibitor The A., Had-Aissouni L. (2008) Preferential vulnerability of mesencephalic dopamine to mesencephalic neurons vulnerabilityof Preferential (2008) Had-AissouniL. A., Nafia I., D.B.,Re Masmejean F., Melon C., Kachidian P., Kerkerian-Le Goff L., Nieoullon dysfunction.glutamate transporter J. Neurol. Sci. Neurol. J. Neuron

90

151 J. Phys. Chem.B. , 675-91. , 83-7. J. Neurochem. J. Mol. Pharmacol. Mol. Ann. Neurol. Ann. nderstanding Dopaminergic J. Neurosci. el P.P. (2003) Prevention of dopaminergicof (2003)Prevention P.P. el ry transmission synaptic via apresynaptic

al/glial mesencephalic cultures requires the the requires cultures mesencephalic al/glial 105

114 , 484-96. ,

58 , 9010-6. ,

64 , 182-93. , , 578-86. 578-86. ,

Curr. Drug Targets 14 , 6754-62. Cell Death Pathways

8 , 621-32 FASEB J. FASEB

27 , Accepted Article This by is protectedarticle reserved. Allrights copyright. inhibition in a cationic pentameric ligand-gated ion channel. ligand-ga inacationicpentameric inhibition Delarue Structural M., basis N. Fourati Colloc'h (2016) T., Z., xenon PrangéSauguet for L., Anaesth. Sanders R.D., Franks MazeN.P., M. (2003) Xenon: nostranger to anaesthesia. animal models of Parkinson's disease. transporter metal Divalent (2008) E.C. Hirsch Sazdovitch Zhao V., L., Garrick L.M., Nuñez M.T., Garrick M.D., Raisman-Vozari R., N., HunotSalazar Mena AlA., J., S.,Prigent homeostasis. neurons dopamine fromvulnerable degene Rousseau Michel P.P., E., E.C. Hirsch (2013) USA, http://imagej.nih.gov/ij/. Maryland, Bethesda, Health, of National Institutes U.S. ImageJ, (1997-2016) Rasband,W.S., cells. glioma ofNMDAActivation receptor ofglutamate influences MMP-2 of activity andproliferation Dalavaikodihalli Nanjaiah Devi N. Aditi K., Fathima Hurmath Ramaswamy (2014) N., P., 55 subthalamicof the nucleus inara BenazzouzPiallat B., A.,Benabid A.L. (1999) Ne hypoxiccortical neurons by noble the gasxenon. in neurotoxicity of (2003)Prevention KoxMüller W.J. J., W., Schmehl P., Blom C., Petzelt metaphase arrest in rat astrocytes. (1999) Xenoninduces Kox M.,W.J. Hafner W., Schmehl G., Taschenberger C., Petzelt

, 71-7.

91 , 709-17. 709-17. , Neurol. Sci. Mol. Pharmacol.

35 , 823-9. ,

84 , 888-98. Life Sci. Life t model of Parkinson'st model disease. Proc. Natl. Acad.Sci. US A.

65 varez-Fischer D., Arredondo M., Duyckaertsvarez-Fischer ArredondoM.,C., D., , 901-13. ration by preserving mitochondrial calciumration bypreserving mitochondrial 1 (DMT1) contributes to neurodegeneration in ted ion channel structural basisstructural xenon for ted ionchannel The iron-binding protectsThe protein lactoferrin Life Sci. Life uroprotective effect of chronic inactivation inactivation chronic of effect uroprotective

72 PLoS ONE , 1909-18. ,

J. Neural. Transm. Suppl. Neural. J. 105 , 18578-83.

11 , e0149795. , Br. J. Br. J.

Accepted Article FASEB J. FASEB This by is protectedarticle reserved. Allrights copyright. after neonatal hypoxia-ischemia. ordelayed long-term provides immediate with xenon inhalation neuroprotection equivalent Chakkarapa HobbsThoresen M., C.E., Wood T., associated with iron accumulation. Fenton reaction in particlessubmitochondrial under oxidative stress: implications for diseases Thomas C., Mackey M.M., Diaz A.A., Cox D.P. 2636-42. M.,Lemaire Kroemer G. (2013)Antiapoptotic activity of argon andxenon. Spaggiari KeppS., O.,Rello-Varona S., Chaba K., Adjemian S., Pype Galluzzi J., L., neuroicells: modeling Application for P.P.R., Michel approach (2016)Asimplified fo S., Raisman-Vozari S., Guerreiro S.B., Hamadat Socias M.O., Ouidja J.E., Sepulveda-Diaz disrupts mitochondrial dopamine aglia midbrain neurons by repressing Verkhratsky A., Kirchhoff F. neuronsprotects against glutamate-inducedexcitotoxicity. (2013) Dopamine Abramov A.Y. S., Gandhi K.M., Holmström Kovac A., S., Vaarmann Toulorge D., Guerreiro S., Hirsch E.C., Michel Hirsch E.C., K PP. Guerreiro S., (2010) Toulorge D., vasodilator, ifenprodil. BDNFof NGF, GDNFand inmouse astrocyte is stronglycultures by a enhanced cerebral ToyomotoInoue M., S., K..,Kuno Hayashi Ohta S, OhtaM., K., Ikeda K. (2005) Production Neuroprotection of midbrain dopamine neurons dopamine midbrain of Neuroprotection A., Hild MaskosGuerreiro S., Toulorge Hirsch D., U., (2011)EC., P.P. Michel

25 , 2563-73. calcium homeostasis. Neurosci. Lett. (2007) NMDAReceptors in glia. J. Cereb. Blood Metab.Flow Redox. Rep. Redox.

nflammatory responses invitro. 379 , 185-9. , J. Neurochem.

14 -to-neuron signaling cascade cascade ultimately signaling that -to-neuron by nicotine is gated by cytoplasmic Ca bycytoplasmic gated is by nicotine r efficient isolation of isolation functional microglialr efficient (2009) Hydroxyl isproducedvia radical the (2009) ni E., Dingley J. (2009)Cooling E., Dingley combined J. ni , 102-8. , Cell Death Dis.

114 29 Neuroscientist ATP , 553-64. , 707-14. , channel blockade protects blockade channel Glia

4 64 , e455.

, 1912-24. 13 Cell Cycle , 28-37.

12 2+ . , Accepted Article This by is protectedarticle reserved. Allrights copyright. subthalamic nucleus in MPTP-treated monkeys. dopaminergic(2007) Survival cells ofmidbrain Wallace B.A., Ashkan HeiseK., Foote C.E., N., K.D., Torres Mitrofanis J., Benabid A.L. Neurochem. Res of [3H]-D-aspar andrelease uptake 2,4-PDC) on beta-benzyloxyaspartate a (DL-TBOA) DLthreo- Schousboe K., Waagepetersen Shimamoto H.S., Neuropsychopharmacology factor release from astroglia andanti-infla Lu mechanis(2009) neuroprotective Novel R.B. Hong J.S., P., Flood S.M., Rawls S.H., Chen Hu X., S.J., Wei L., Qian Tzeng N.S., Wu H.M., factor (erythroid-derived 2)-like 2. 2)-like (erythroid-derived factor hypoxic-ischemic againstprotects injury brain neonatalrats through nuclear in of activation Zhao Mitchell S., H., Ciechanowicz S., Savage JiWang S., Ma T., X., (2016) D. Argon American Zhang and XuZ. (1995) radii of Y., Atomic gas noble elements in condensed phases. PLoS One protection brain and fetal duringanalgesia labor provide sevoflurane Ma M., Maze (2012)Xenon and Robertson N.J., Johnson D. M..R, R.D., Sanders WuN., X., E., Terrando Petrides A.M., Fidalgo ZhuangL., Rei Yang T., Anaesthesiol. Scand. with on ischemic spinal comparison of xenon propofol. a inrabbits: cord injury (2010)Effects H. S., Furuya Inoue M., Kakimoto N., Kurita Kawaguchi M., Y., Yamamoto

Mineralogist 7 , e37020. . 26 , 661-6. 54 , , 337–342. 337–342. , 80 , 670-675,

34 , 2344-57. Oncotarget mmation activation. by microglial preventing 7 , 25640-51. , Brain in a perinatal in rat aperinatal model of hypoxia-ischemia. after ordeeplesion after stimulation brain of the nd L-trans-pyrrolidine-2,4- dicarboxylate (t- nd L-trans-pyrrolidine-2,4- dicarboxylate tate inastrocyte andglutamatergictate neurons. ms of memantine: increase in in neurotrophic memantine: ms of increase A. (2001) Comparison of effects of effects of (2001) Comparison A. 130 , 2129-45. Acta Acta Accepted Article N treated ornot withcultures PDC24 hours gas for under atmospheres 75%containing either DA neurons PDC-treated FigureNMDA2. receptor antagonismaccounts thefor rescuing effectxenon of for days underanatmosphere containing 75%N control cultures under N under control cultures cultures maintained under N under maintained cultures maintained under N maintained under (100 exposed or not to PDC(100 not to or exposed 100 control N 75% Comparison with withXe. 10 memantinetreatment a (MEM, Xe for midbrain TH midbrain for Xe ( atmosphere. cultures underN cultures ±S.E.M.( mean indicate bars Error Ar. or 75% This by is protectedarticle reserved. Allrights copyright. ofrate Survival DAneurons (TH PDC. by deathinduced DAagainst cell rescue partial Xenon provides 1. Figure Figure legends a same concentration N of PDC under cultures maintained under N cultures ( Xe. Error bars indicate mean ±S.E.M. indicate mean Errorbars Xe. n 2 = 16). or 75% Xe. Error bars indicate mean mean or 75% ±S.E.MError bars Xe. indicate *** (n = 9). μ μ M PDC for 1( M PDCfor M) or NMDA (75µM) for for 4 days underan atmosphere containing either 75%N M) orNMDA (75µM)

*** 2 atmosphere. Error bars indicatemean ±S.E.M. ( P <0.001 relative to control cultures cultures and control to relative <0.001 d ) Inverted fluorescence) Inverted images illustrating neuroprotective the effectsof 75% 2 atmosphere and atmosphere + neurons exposed 1or for 4days to 100 n 2 =10) or4days ( atmosphere and atmosphere 2 atmosphere and and atmosphere μ . 2 (a M) days for anatmosphere 4 containingunder 75%N atmosphere. ( atmosphere. 2 atmosphere. ( ) Extracellular levels of glutamate measuredExtracellular midbrainof levels) glutamate in + cells) incultures(10-300 not to exposed cells) PDC or ### 2 n P ***P atmosphere. ( atmosphere. <0.001PDC-treated cultures under to relative N = 16) in an atmosphere containing= 16)inanatmosphere 75%N ### b P <0.001 relative to age-matched control cultures cultures to age-matched control relative <0.001 ) Survival rate of DA neurons exposed to PDCDA neuronsexposedto of rate Survival ) 2 ### <0.001 to relative age-matched PDC-treated or 75% Xe. Error bars indicate ±S.E.M.mean indicate bars or Error 75% Xe. c ) Survival rate of DA neurons in cultures P <0.001 relative to corresponding cultures cultures tocorresponding relative <0.001 n = 16). 16). = ### b P ) Survival of DA of neuronsto) Survival exposed <0.001 relative to cultures receiving receiving to cultures relative <0.001 n = n = μ ***P M ofPDC. Scale bar:40 6-9) P < 0.001 relative to control to relative < 0.001 <0.001 relative to control<0.001 relative .

μ ***P M) performed under a under M) performed <0.001 relative to relative <0.001 2 μ , 75% Xe 75% , 2 M) for 4 M) for or75% μ m. m. 2 or ( a 2 )

Accepted Article atmosphere atmosphere containing 75%N an under 10µM) (DESF, ordesferioxamine 10µM) Trolox(TROL, not concomitantly with Survival of DA neurons in 4 cultures exposedfor daysor not andtreated to PDC or(100 µM) (MEM, 1- 30 (MEM, to PDC (100 indicate mean ±S.E.M. ( cultures maintained under75%N cultures This by is protectedarticle reserved. Allrights copyright. maintained undercultures N 75% § # N under PDCNMDA, toeither or exposed atmospheres containing either 75%N 4 presence days Trolox various(0.1-10 in the concentrations of or notof DHR-123 in midbrain cultures maintained cultures DHR-123 under inmidbrain 75%N probe of fluorescent intracellular the (c)Quantification with ROSTrolox. production of under 75% Xe and PDC-treated cultures maintained under 75%N culturesunder 75%XeandPDC-treated maintained under Figure3. Xenon reduces the impact of 40 bar Scale 4days. for 10 + (MEM, memantine images that fluorescence showing impact the relative to control cultures under 75%N under tocontrol cultures relative barsindicate mean±S.E.M. Error atmosphere containing 75%Xe. ( treated cultures under N *** P P <0.05 relative under75%Xe PDC-treated maintained and<0.05 toPDC-treated cultures to <0.05, P <0.001 relative to control cultures under N cultures tocontrol relative <0.001 ## P μ <0.01 and <0.01 and M) for 4 days in the presence, or not, of various concentrations of memantine of various concentrations ornot, of the presence, 4days in for M) μ M) undergas atmospheres containing 75%N either 2 μ n ### atmosphere. m. m. μ = 9). M) exert on the survival of DA neurons exposed to 100µM PDC DAneuronsexposedto100µM survival of onthe exert M) P <0.001 relative to PDC-treated culturesunder N PDC-treated relative to <0.001

*** 2 2 . Comparison . to cultures exposed to an PDC under . (b) Survival of DA neurons exposed to PDC(100 neurons exposed DAof Survival (b) . 2 P and a same concentration of memantine. ( <0.001 relative N control culturesunder <0.001 to 2 §§§ or 75% Xe. Error bars indicate mean ±S.E.M75% Xe. barsindicatemean Error or ( oxidative insults in PDC-treated DA inPDC-treated insults oxidative P 2 and and <0.001 relative to PDC-treated cultures maintained cultures PDC-treated to relative <0.001 2 atmosphere. ( atmosphere. treatments with 75% xenon alone or 75% xenonor 75% alone 75% xenon treatments with # P <0.05 or <0.05 2 . # P <0.05 and and <0.05 2 c ### or 75% Xeand 1day treated for ) Survival of DAneuronsexposed) Survival of P <0.001 relative PDC-treated to ## 2 and a same concentrationsame a and P 2 n <0.01 relative to PDC- or 75% Xe. Error bars Errorbars or75% Xe. = 11-13). *** =11-13). μ M) under gas under M) 2 2

atmosphere. atmosphere. neurons. d ) Inverted ) Inverted P μ <0.001 n M) for = 9). (a) (a)

Accepted Article ±S.E.M. (n = 9). * presence of memantine. ±S.E.M. (n = 9). * memantine 10 or not or of (MEM, presence 75% Xeinthe memantine (MEM, 10 µM) and maintained under 75% N 75% maintained under and 10µM) memantine (MEM, or 7-14 10µM), (TROL, DA between neuronsDIV Trolox Survival treated with of relative culturesrelative to maintained under 75% containing 75% N an atmosphere under maintained cultures DIV midbrain and 14 in 7 glutamate concentrations This by is protectedarticle reserved. Allrights copyright. 75% N under maintained cultures control ±S.E.M. ( indicate mean bars Error 10µM). memantine (MEM, or 0.1 and10µM) inPDC, (TROL, withTrolox alone orcombination with 100µM * meanindicate ±S.E.M. (n=9). Error bars time interval. same 75% Xe during the to exposed 9). * 9). ba Error supplementedcultures withserum. NMDA receptor antagonism. Figure 4. Xenon to spontaneously protection neurons dying DAprovides through PDC-treatedproduction in Scalebar: 40µm. cultures. DA neurons maintained 7-14DIVunder between DA neurons maintained maintained under 75% N atmosphere atmosphere containing 75%N cu DAneuronsin of Survival (b) cultures. relative to control cultures after plating, i.e., atDIV.plating, 0 i.e., cultures after tocontrol relative P <0.05 relative to control cultures maintained maintained under 75%N cultures relative to <0.05 control P <0.05 relative to control cultures maintained under N under cultures maintained to control relative <0.05 2 P and in 14 DIV keptcultures under75%Xe. Errorbars indicate mean P <0.05 relative <0.05 underto 7 DIV maintained relative cultures N <0.05 relative to control cultures under 75% N cultures under 75% to control relative <0.05 2 . (d)Digitized images. theimpact illustrating of 75%Xe on ROS 2 (a) Survival of DA neurons at DIV 0, 7 and 14 in midbrain midbrain 7and14in 0, DA neuronsat DIV of (a)Survival , 75% Xe or 75% Ar. Error bars indicate mean ±S.E.M. Error barsindicate mean (n= 75%XeorAr. , 2 and, and, Xe cultures and maintained under 75%N ltures maintained between maintained 7-14 ltures DIV underan rs indicate mean * ±S.E.M.= 6). rs indicate (n ### gas atmospheres containing 75%N either gas atmospheres P <0.001 relative toPDC-treated cultures # P <0.05 relative to 7 DIV control 2 μ atmosphere. (c) Extracellular n 2 2 M). Error bars indicate meanbars indicate Error M). . Comparison. withcultures atmosphere. (e) Survival of of Survival (e) atmosphere. = 6). *** 2 atmosphere. atmosphere. P <0.001 relative to relative <0.001 2 atmosphere. (d) atmosphere. § 2 P P in the <0.05 <0.05 <0.05 <0.05 2

Accepted Article cultures receiving the same treatments in treatments as same receiving the cultures memantine. (c)Measurement of total neurite atmosphere atmosphere containing 75%N an under 10µM) memantine (MEM, without or with days 7 treatedfor cultures midbrain (TH maintained under 75% Xe and cultures maintained under 75% N under maintained cultures Xe and 75% maintained under Data are means ±SEM (n=9). previous onall treatments. Impactatmosphere. of a GDNF antibody neutralizing (10µg/mL) kept underacontrolin cultures Comparison with GDNF treatment (20ng/mL) 10µM). for 7 days under 75% 7daysfor N under astrocytes. Figure 6. Xenon rescues DAspontaneously dying neurons when treatments are used, separately,or in combination. Scalebar 20 and the xenon haverepresentative DA the memantine impact on morphologythat of of relative to corresponding treatments without GDNFcorresponding without to relative antibody treatments relative tomemantine-treated cultures under N This by is protectedarticle reserved. Allrights copyright. 75% cultures maintained14 DIVDA under neuronsin Survivalmidbrain memantine. (b) of neurons. DA spontaneously dying of differentiation cooper act memantine Xenon and 5. Figure means ±SEM (n=9). * (n=9). ±SEM means to control cultures under 75% N under 75% cultures control to maintained under 75% Xe and cultures maintained under 75% N 75% under maintained cultures and Xe 75% maintained under cultures maintained undercultures 75% N 0.05 relative to relative75% N 0.05 under to control cultures + ) neuron in14 DIV cultures) neuron midbrain receiving thesame treatments as in(a). Data are (a) Survival(a) DA of neurons in 14 DIV cultures midbrain previously maintained P <0.05 relative to control cultures under N cultures tocontrol relative <0.05 2 or 75% Xe, in the presence or absence of(MEM,presence memantine or absence the in or 75% Xe, 2 or 75% Xe. Data are means ±S.E.M. (n=9). * *P 2 . <0.05 relative to control cultures under cultures 75%N relative <0.05 to control § 2 P in the presence of memantine. (b) DA uptake per(b) DA of memantine. presence in DAuptake the <0.05 relative to cultures maintained under 75% Xe and relative tocultures maintained 75%Xe <0.05 under (a) and (b). Data are means ±SEM (n=9). * ±SEM means (n=9). are Data (b). and (a) 2 density perDA neuron in 14 DIV midbrain atmosphere. (d) Inverted fluorescence images images fluorescence (d) Inverted atmosphere. 2 atmosphere. atively atively to improve and the function neurons through a repressive effect on effect arepressive through neurons (a) Uptake of [ of Uptake (a) . § § P P <0.05 relative to cultures cultures to relative <0.05 <0.05 relative to cultures to relative <0.05 2 atmosphere. 2 2 in the presence of presence the in in the presence of 3 H]-DA in 14DIV H]-DA in P μ m. <0.05 relative<0.05

2 . # # P P <0.05 <0.05 <0.05 <0.05 P < Accepted Article

* ±SEM (n=15). means are Data (b). in same treatments as receivingthe cultures DIV midbrain control cultures under75% N control cultures * ±SEM(n=9). means are Data 3 DIV. for (c) and (b) in as treatments same and the same non-gaseous treatment. (c) Quantification of [ (c) Quantification same non-gaseous treatment. and the (10 µM). Data are means ±SEM (n=9). * ±SEM(n=9). means are Data (10 µM). This by is protectedarticle reserved. Allrights copyright. cultures undermaintained 75% N § N (d) Quantification of [ of (d) Quantification under 75% Xe and under 75%Xe

P P 2 <0.05 <0.05 relative to cultures maintained under 75% Xe and cultures maintained under 75% N or 75% Xe for 7 days in the presence or thepresence in 7days Xe for or75% < 0.05 relative to control cultures under 75%N under cultures tocontrol relative 0.05 <

cultures maintained under 75% N maintained cultures 3 H]-thymidine H]-thymidine incorporation inpure astrocyte receivingcultures the 2 . § 2 P and and samethe non-gaseous treatment. <0.05 relative maintained tocultures and under 75%Xe P < 0.05 relative to control cultures under 75%N cultures under tocontrol relative < 0.05 absence of memantine (MEM, 10µM) or AraC or 10µM) memantine(MEM, of absence 2 . § 2 P and the non-gaseous same and treatment. <0.05 relative to cultures maintained 3 H]-thymidine incorporation in 14 in incorporation H]-thymidine P <0.05 relative to relative <0.05 2 2 .

Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.