ARTICLES. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals

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1 7 Ntur Pulishing Group yrogn cts s thrputic ntioxint y slctivly rucing cytotoxic oxygn ricls Ikuroh Ohsw 1, Mshiro Ishikw 1, Kumiko Tkhshi 1, Mgumi Wtn 1,, Kiyomi Nishimki 1, Kumi Ymgt 1, Kn-ichiro Ktsur, Ysuo Ktym, Smitsu soh 1 & Shigo Oht 1 cut oxitiv strss inuc y ischmi-rprfusion or inflmmtion cuss srious mg to tissus, n prsistnt oxitiv strss is ccpt s on of th cuss of mny common isss incluing cncr. W show hr tht hyrogn ( )hs potntil s n ntioxint in prvntiv n thrputic pplictions. W inuc cut oxitiv strss in cultur clls y thr inpnnt mthos. slctivly ruc th hyroxyl ricl, th most cytotoxic of rctiv oxygn spcis (ROS), n ffctivly protct clls; howvr, i not rct with othr ROS, which possss physiologicl rols. W us n cut rt mol in which oxitiv strss mg ws inuc in th rin y focl ischmi n rprfusion. Th inhltion of gs mrkly supprss rin injury y uffring th ffcts of oxitiv strss. Thus cn us s n ffctiv ntioxint thrpy; owing to its ility to rpily iffus cross mmrns, it cn rch n rct with cytotoxic ROS n thus protct ginst oxitiv mg. Oxitiv strss riss from th strong cllulr oxiizing potntil of xcss rctiv oxygn spcis (ROS), or fr ricls 15.Mostofth suproxi nion ricl (O ) prouc is gnrt in mitochonri y lctron lkg from th lctron trnsport chin n th Krs cycl 6.O is lso prouc y mtolic oxiss, incluing NDP oxis n xnthin oxis 7. Suproxi ismuts convrts O into hyrogn proxi ( O ) 8, which is toxifi into O y ithr glutthion proxis or ctls. Excss O rucs trnsition mtl ions such s F 3 n Cu (rf. ), th ruc forms of which in turn cn rct with O to prouc hyroxyl ricls ( O) y th Fnton rction. O is th strongst of th oxint spcis n rcts iniscrimintly with nuclic cis, lipis n protins. Thr is no known toxifiction systm for O; thrfor, scvnging O is criticl ntioxint procss 9. Dspit thir cytotoxic ffcts, O n O ply importnt physiologicl rols t low concntrtions: thy function s rgultory signling molculs tht r involv in numrous signl trnsuction cscs n lso rgult iologicl procsss such s poptosis,cll prolifrtion n iffrntition 7,1.thighrconcntrtions, O is convrt into hypochlorous ci y myloproxis; hypochlorous ci fns ginst ctril invsion 5.Nitricoxi(NO ), nothr ROS, functions s nurotrnsmittr n is ssntil for th iltion of loo vssls 11.Thus,cytotoxicriclssuchs O must nutrliz without compromising th ssntil iologicl ctivitis of othr, physiologiclly nficil, ROS. r w monstrt tht molculr hyrogn (ihyrogn, ) cn llvit O-inuc cytotoxicity without ffcting th othr ROS, n propos tht hs potntil s n ntioxint for prvntiv n thrputic pplictions. RESULTS slctivly rucs O in cultur clls rucs th O tht is prouc y riolysis or photolysis of wtr 1 ; howvr, whthr cn ffctivly nutrliz O in living clls hs not n irctly invstigt. s th cllulr mg prouc y spontnous gnrtion of O is not sufficint to tctl, w inuc O prouction in PC1 cultur clls. To o this, w trt th clls with mitochonril rspirtory complx III inhiitor, ntimycin (rf. 13); following such trtmnt, O in ths clls is rpily convrt into O. Th ition of ntimycin incrs lvls of O n O, s jug y th fluorscnc signls mitt y th oxiiz forms of MitoSOX (Fig. 1) n,7 -ichloroihyrofluorscin ( DCF) (Supplmntry Fig. 1 onlin), rspctivly. W issolv n O into mium s scri in th Mthos, n confirm th prolong (4 h long) mintnnc of lvls (Supplmntry Fig. onlin). issolv in cultur mium i not crs MitoSOX n DCF signls in th clls (Fig. 1, n Supplmntry Fig. 1). itionlly, i not crs th sty-stt lvl of NO (Supplmntry Fig. 1). In contrst, trtmnt significntly crs lvls of O, s ssss y th fluorscnc signl mitt y th oxiiz form of -[6-(4 -hyroxy)phnoxy-3-xnthn-3-on-9-yl] nzot (PF) (rfs. 14,15 n Fig. 1c,). Whn w xpos th clls to ntimycin (3 mg/ml) in th snc of, th PF signls incrs in oth th nuclr rgion n th cytoplsm, proly cus O iffus from th mitochonri to prouc O. Notly, crs O lvls vn in th nuclr rgion (Fig. 1c). 1 Dprtmnt of Biochmistry n Cll Biology, Institut of Dvlopmnt n ging Scincs, Grut School of Micin, Nippon Micl School, Kosugi-cho, Nkhr-ku, Kwski City , Jpn. Dprtmnt of Intrnl Micin, Nippon Micl School, Sngi, Bunkyo-ku, Tokyo , Jpn. Corrsponnc shoul rss to S.O. (oht@nms.c.jp). Rciv Sptmr 6; ccpt 15 Mrch 7; pulish onlin 7 My 7; oi:1.138/nm VOLUME 13 [ NUMBER 6 [ JUNE 7 NTURE MEDICINE

2 7 Ntur Pulishing Group c Figur 1 Molculr hyrogn issolv in mium slctivly rucs hyroxyl ricls in cultur clls. (,) PC1 clls wr incut in mium with or without.6 mm, n xpos to ntimycin (3 mg/ml for 3 min) in orr to inuc O prouction. Thy wr thn trt with.5 mm MitoSOX. Rprsnttiv fluorscnc imgs of MitoSOX-trt clls wr otin y lsr-scnning confocl microscopy (Olympus FV3). MitoSOX fluorscnc ws quntifi from 1 clls of ch inpnnt xprimnt (n ¼ 5). (c) Rprsnttiv lsr-scnning confocl imgs of th fluorscnc of th O mrkr PF wr tkn 3 min ftr th ition of ntimycin. rrows n rrowhs inict th incrs n crs, rspctivly, in PF signls in th nuclr rgion. () PF fluorscnc in clls trt with ntimycin with or without.6 mm ftr ntimycin trtmnt, prvnt th clin of th mitochonril mmrn potntil, s tct y fluorscnc of ttrmthylrhomin mthyl str (TMRM), which pns upon th mitochonril mmrn potntil, whrs fluorscnc lvls of MitoTrckr Grn (MTGrn), which r inpnnt of th mmrn potntil, wr unchng (Fig. 1). This suggst tht protct mitochonri from O. -trt clls look norml, whrs -untrt clls wr shrunkn n h norml roun shps (Fig. 1). long with this protctiv ffct, lso prvnt crs in th cllulr lvls of TP synthsiz in mitochonri (Fig. 1f). Th fct tht protct mitochonri n nuclr DN provi vinc tht pntrt most mmrns n iffus into orgnlls. issolv in mium protcts cultur clls ginst O W plc PC1 clls in cultur mium contining n O,n, t th sm tim, inuc oxitiv strss y ing ntimycin. t 4 h ftr th inuction of ROS with ntimycin, w osrv tht sm to protct nuclr DN from oxition, s shown y crs lvls of oxiiz gunin (8-O-G) (Fig., n rf. 16). Morovr, lso crs lvls of 4-hyroxyl--nonnl (NE), n n-prouct of lipi proxis (Fig. c, n rf. 17), inicting tht it protct lipis from proxition. Furthr, issolv in mium protct clls from cll th in os-pnnt mnnr (Fig.,f). Whn w rmov from mium tht h n sturt with, th protctiv ffct isppr (Fig. f), suggsting tht th osrv ffct ws not u to rction of with th mium. Morovr, w confirm tht protct cllulr viility y using two mthos: moifi MTT ssy (WST-1 ssy) n msurmnt of cllulr lctt hyrogns (LD) lkg from mg clls (Supplmntry Fig. 3 onlin). To xclu th possiility tht th protctiv ffct of ws u to rction with Fluorscnt intnsity pr cll 1 3 MTGrn TMRM Mrg ntimycin, w inuc ROS y ing mnion, n inhiitor tht cts on mitochonril complx I, n osrv tht protct clls in this systm s wll (Supplmntry Fig. 3). To vrify tht protcts ginst O, w prtrt clls with Cu n thn xpos thm to scort, in orr to ruc intrcllulr Cu to Cu, which in turn ctlyzs th prouction of O from cllulr O tht is nognously prouc. This trtmnt primrily inuc O insi th clls (y th Fnton rction), thus irctly confirming tht protcts clls ginst cllulr O (Fig. g,h). Spin-trpping intifis fr ricl tht is ruc y To intify th fr ricl spcis tht rucs, w stui th ffcts of on lctron spin rsonnc (ESR) signls of spin-trpping rgnts. W prouc O y th cllulr Fnton rction, n smiquntifi th cllulr lvls of O y spin-trpping using 5,5-imthyl-1-pyrrolin N-oxi (DMPO). Msurmnts of ESR inict tht trtmnt i in crs signls of DMPO-O riv from O (Fig. 3c). Morovr, whn w inuc O prouction y trting clls with ntimycin in th prsnc of DMPO, w osrv multipl ESR signls 18. Ths signls sm to consist of thos from th DMPO- O n DMPO- ricls (Fig. 3f). Th DMPO- ricl is riv from th hyrogn ricl ( ), which cn inuc y porphyrins. To visuliz th signls crs y, w otin th iffrntil spctrum. W foun tht only O-riv signls wr crs y trtmnt (Fig. 3). Ths rsults strongly suggst th slctiv ruction of cllulr O y trtmnt. slctivly rucs O n ONOO in cll-fr systms Nxt, w confirm in pur solution tht PF fluorscnc cn us to monitor th ruction of O y uring continuous O Fluorscnt intnsity pr cll f Rltiv TP lvl (%) h 1 h ws quntifi from 1 clls (n ¼ 4). P o.1, P o.1. () t 3 min ftr ing ntimycin (1 mg/ml) with or without (.6 mm), clls wr incut with 1 mm MTGrn n 1 nm TMRM for 1 min n thn img. Th two imgs wr suprimpos (mrg). (f) Clls wr prtrt with 4.5 g/l -oxyl-d-glucos (n inhiitor of glycolysis) n 1 mm pyruvt, n rltiv cllulr TP lvls wr quntifi ftr xposur to 3 mg/ml ntimycin. TP lvls of clls not trt with ntimycin wr st t 1% (n ¼ 3). P o.5, P o.1. Scl rs: 1 mm in; 5mm inc; mm in. istogrms show mn ± s.. NTURE MEDICINE VOLUME 13 [ NUMBER 6 [ JUNE 7 689

3 7 Ntur Pulishing Group No. of surviving clls pr FOV 16 8 ntioy to 8-O-G 8-O-G (pixl intnsity pr cll) issolv (µm) ntimycin Dgss ochst 3334 PI 1 1 NE-protin conjugts (pixl intnsity pr cll) ntimycin f 3 g Cu h Vit. C 4 4 ntioy to NE c Cll th (%) Vitmin C (µm) Figur Molculr hyrogn protcts cultur PC1 clls y scvnging hyroxyl ricls. () PC1 clls wr mintin with 1 mg/ml ntimycin, with () or without ().6 mm, for 4 h in clos flsk, n immunostin with ntiois to 8-O-G or NE. Fluorscnc signls in rspons to 8-O-G n NE wr quntifi using 1 clls from ch inpnnt xprimnt (n ¼ 4). P o.5, P o.1. () Phs-contrst picturs of PC1 clls 4 h ftr th xposur to ntimycin, with () or without ().6 mm. rrows inict clls. (f) Cll survivl ws ssss y mnully counting th clls (Mthos; n ¼ 4). P o.5, P o.1 (compr with O mm ). (g) PC1 clls wr xpos to intrcllulr O prouc y th Fnton rction, with or without.6 mm. Clls wr princut with 1 mm CuSO 4, wsh, n xpos for 1 h to.1 mm scort (Vit. C) in orr to ruc intrcllulr Cu to Cu (Supplmntry Mthos). Th clls wr costin with propiium ioi (PI) (for clls) n ochst 3334 to visuliz th nucli. (h) Cll survivl ws ssss y mnully counting th clls s in f (n ¼ 5). P o.5, P o.1. Scl rs: 5 mm in,c,; 1 mm in g. istogrms rprsnt mn ± s.. prouction y th Fnton rction. In this conition, supprss incrss in PF signls in os-pnnt mnnr (Fig. 4c). But whn w mix solution contining with PF proxiiz with O, fluorscnc signls from oxiiz PF i not crs (t not shown), supporting th i tht irctly rcts with O. Nxt, w xmin th rctivity of with othr ROS or rctiv nitrogn spcis (RNS). W prpr O n proxynitrit (ONOO ) y ilution of th rspctiv stock solutions, O y th nzymtic rction of xnthin oxis with xnthin, n NO y th spontnous rction of 1-hyroxy--oxo-3-(N-mthyl-3-minopropyl)-3-mthyl-1-trizn (NOC7) in cll-fr systms (Supplmntry Mthos onlin). ruc ONOO (Fig. 4) somwht, ut i not ruc O,NO n O (Fig. 4g). In cll-fr xprimnts, w xmin whthr ruc th oxiiz forms of iomolculs involv in mtolic oxition-ruction rctions. t room tmprtur n nutrl p, solutions sturt with i not ruc th oxiiz form of nicotinmi nin inucloti (ND ), th oxiiz form of flvin nin inucloti (FD) or th oxiiz form of cytochrom C (t not shown). Thus w infr tht os not ffct th mtolism involv in oxition-ruction rctions or th lvls of O, O, n NO, ll of which ply ssntil rols in signl trnsuction. protcts nurons from in vitro ischmi n rprfusion W lso inuc oxitiv strss in primry cultur of nocorticl clls 19 unr mor physiologicl conitions. It is known tht rpi trnsition from n ischmic conition to rprfusion rsults in oxitiv strss mg. To mimic ischmi, w sujct nocorticl clls to oxygn glucos privtion (OGD) unr nitrogn or hyrogn gs for 6 min, follow y rprfusion with mium contining O n glucos. PF fluorscnc show tht 1 min ftr th compltion of OGD follow y rprfusion, O lvls notly incrs whn ws snt, ut iminish whn ws prsnt (Supplmntry Fig. 4 onlin). t 4 h ftr OGD n rprfusion, incrs nuron survivl n vitlity (Supplmntry Fig. 4), inicting tht protcts nurons ginst oxitiv strssinuc cll th. Inhltion of gs protcts rin injury y rprfusion To xmin th thrputic pplicility of s n ntioxint, w us rt mol of ischmi. ROS r gnrt uring crrl 69 VOLUME 13 [ NUMBER 6 [ JUNE 7 NTURE MEDICINE

4 7 Ntur Pulishing Group c Mn Mn G, Fnton rction, Fnton rction ischmi n rprfusion, n r on of th mjor cuss of rin mg 1,. W prouc focl ischmi in rts y occluing th mil crrl rtry (MC) for 9 min, n thn prform rprfusion for 3 min (rf. 3). In thr of four conitions, rts inhl gs, mix with nitrous oxi (N O) for nsthsi, uring th ntir 1 min procss (proportions of,o n N O(vol/vol/ vol) wr 1%:3%:69%, %:3%:68%, n 4%:3%:66%); in th fourth conition, ws snt, ( :O :N O (vol/vol/vol) ws %:3%:7%). W crfully monitor physiologicl prmtrs uring th xprimnts (Mthos) n foun no significnt chngs rsulting from th inhltion of (Supplmntry Tl 1 onlin). itionlly, thr ws no significnt influnc on crrl loo flow, f g C 3 C 3 C 3 C 3, ntimycin G, ntimycin γ α β N O γ α β N O O Figur 3 Spin-trpping intifis th fr ricl spcis tht rucs. () Stnr lctron spin rsonnc (ESR) signls of th DMPO- O ricl wr otin y trpping O with spin-trpping rgnt (DMPO; tils in Supplmntry Mthos). (,c) PC1 clls wr princut with.1 M DMPO n mm CuSO 4 for 3 min t 37 1C with or without.6 mm. ftr rmovl of this mium, th clls wr trt with. mm scort n.1 mm O for 5 min t 3 1C to prouc O n thn scrp into flt cuvtt for ESR msurmnt. Rctngl hight rflcts signl intnsity. () Th DMPO-O n DMPO- ricls 18 n thir corrsponing ESR signls r illustrt. (,f) PC1 clls wr incut in PBS contining.1 M DMPO n 3 mg/ml ntimycin for 7 min t 3 1C, with or without.6 mm, thn scrp into flt cuvtt for ESR msurmnt. (g) iffrntil spctrum ws otin y sutrcting th spctrum in f from tht in, in orr to visuliz th signls crs y trtmnt. inicts DMPO-O signls riv from O. n o inict DMPO-O n DMPO- signls, rspctivly (,,, n g). s msur y th Dopplr ffct (rf. 4 n Supplmntry Fig. 5 onlin). issolv in rtril loo ws incrs y th inhltion of in proportion to th concntrtion inhl; th mount of issolv in vnous loo ws lss thn tht in rtry loo, suggsting tht h n incorport into tissus (Fig. 5). t 1 ftr MC occlusion, w sction n stin rins with,3,5-triphnylttrzolium chlori (TTC), sustrt for mitochonril rspirtion (Fig. 5). W stimt infrct volums y ssssing th stining of rin rs (whit inicts infrct, Fig. 5,c), n foun clr -pnnt crs in infrct volum, with 4% of proviing th most sustntil ffct (Fig. 5c). W lso not tht xrt its ffct only whn it ws inhl uring rprfusion; whn ws inhl uring ischmi, infrct volum ws not significntly crs (Fig. 5,). For comprison, w tst two othr compouns: rvon (pprov in Jpn s n ROS scvngr for th trtmnt of crrl infrction ) n FK56 (in clinicl trils for crrl infrction in th Unit Stts 6 ). ws mor ffctiv thn rvon n s ffctiv s FK56 in llviting oxitiv injury (Fig. 5c). Ths rsults inict th potntil of for thrpy. Inhltion of gs supprsss th progrssion of mg t 1 wk ftr MC occlusion, th iffrnc in infrct volum twn untrt n -trt rts incrs, compr to 1 ftr Fluorscnt intnsity (ritrry units) mm. mm.4 mm.8 mm Bslin 1 Bslin Tim (s) Initil vlocity (ritrry units) concntrtion (mm) c f Fluorscnt intnsity of PF (%) (1 µm) O ONOO O O NO 14 Fluorscnt intnsity of PF (%) (1 µm) sornc of NBT-iformzn (%) SOD (1 units) Fluorscnt intnsity of PF (%) (1 µm) g Fluorscnt intnsity of DF- (%) (1 µm) Figur 4 Molculr hyrogn issolv in solution scvngs hyroxyl ricls t 3 1C n p 7.4 in cll-fr systms. (,) Th Fnton rction, which gnrts hyroxyl ricls, ws initit y ing O (to finl concntrtion of 5 mm) in clos cuvtt t 3 1C with gntl stirring (Supplmntry Mthos). Lvls of O in th prsnc of vrious concntrtions of issolv in th solution wr ssss for PF fluorscnc. () Rprsnttiv tim cours trcs of PF fluorscnc t ch concntrtion of. Bslins 1 n show PF fluorscnc (in th prsnc of.8 mm ) in th snc of O (slin 1) n in th snc of frrous prchlort (slin ). () Mn ± s.. of initil rts of incrs in PF fluorscnc (four inpnnt xprimnts). (cg) Lvls of O n two rctiv nitrogn spcis (RNS: NO n proxynitrit (ONOO )) rmining ftr incution with.6 mm of t 3 1C (tilsinthsupplmntry Mthos). Vitmin C (Vit. C) n suproxi ismuts (SOD) wr us s positiv controls. Signls gnrt in th snc of () wr st t 1%. Dt rprsnt mn ± s.. (n ¼ 6). P o.5, P o.1. NBT-iformzn: oxiiz form of nitrolu ttrzolium (NBT, tctor of O ). DF-: iminofluorscin- ( tctor of NO). NTURE MEDICINE VOLUME 13 [ NUMBER 6 [ JUNE 7 691

5 7 Ntur Pulishing Group Durtion of gs inhltion B C % in loo (ng/ml) MC (9 min) 4 % V 4 c Infrct volum (mm 3 ) 14EF 14EF 14EF occlusion (Fig. 6,). Th hvior of ch rt, gr ccoring to nurologicl scor 7, rvl tht th inhltion of uring ischmi n rprfusion improv movmnt (Fig. 6c). Morovr, lthough oy wight n oy tmprtur of -untrt rts grully clin, thos in -trt rts vntully rcovr (Fig. 6,). Thus supprss not only th initil rin injury, ut lso its progrssiv mg. W xmin -mit molculr chngs t 1 h, 3 or 7 ftr occlusion, y stining rin sctions with ntiois to 8-O-G in orr to ssss th xtnt of nuclic ci oxition (Supplmntry Fig. 6 onlin), n with ntiois to NE to ssss lipi proxition (Supplmntry Fig. 6). For oth of ths oxitiv mrkrs, stining ws sustntilly ruc in -trt rts s compr to untrt rts. W lso stin inticl rgions of th rin with ntiois to I1 (rf. 8) n ntiois to GFP, which r spcific to ctivt microgli n to strocyts, rspctivly (Fig. 6f,g n Supplmntry Fig. 6). W foun istinct -pnnt crs in th ccumultion of microgli, inictiv of inflmmtion n rmoling. Tkn togthr, ths rsults inict tht cn mrkly crs oxitiv strss n supprss rin injury Rprfusion 1 (3 min) min 5 Lst 35 min First 85 min (%) V Infrct volum (mm 3 ) ## ### Stritum Cortx Totl V (%) (%) (%) (%) ### Stritum Cortx Totl # BC BC BC ### Figur 5 Inhltion of hyrogn gs protcts ginst ischmi-rprfusion injury. () Rts inhl n 3% O for 1 h unr th nsthtics N O n hlothn. rtril () n vnous (V) loo wr collct, n th mount of ws xmin y gs chromtogrphy. () Rtsunrwnt mil crrl rtry (MC) occlusion. During th 1-min procur, th inict concntrtion of mix gs ws inhl. On y ftr MC occlusion, th forrin ws slic into six coronl squntil sctions n stin with th mitochonril rspirtory sustrt TTC. Scl r, 5 mm. (c) Infrct volums of th rin wr clcult in th rin slics. E n F, trtmnt with rvon n FK56 (n ¼ 6). P o.5, P o.1, P o.1, compr with % of. ## P o.1, ### P o.1 compr with % of.() Schmtic of xprimnt with thr iffrnt urtions of hyrogn gs (%) inhltion. () Infrct volums of th rin for iffrnt urtions of inhltion (clcult s in c) (n ¼ 6). P o.5, P o.1, P o.1, compr with % of. # P o.5, ### P o.1 compr with 1 min of trtmnt., B n C rprsnt th iffrnt urtions of gs inhltion (shown in ). istogrms rprsnt mn ± s.. DISCUSSION This stuy shows tht molculr hyrogn cn slctivly ruc ROS in vitro. s O n ONOO r much mor rctiv thn othr ROS (rf. 14), it stns to rson tht will rct with only th strongst oxints. This is vntgous for micl procurs, s it mns tht th us of shoul not hv srious unwnt si ffcts. It is likly tht is mil nough not to istur mtolic oxitionruction rctions or to isrupt ROS involv in cll signling unlik som ntioxint supplmnts with strong ructiv rctivity, which incrs mortlity, possily y ffcting ssntil fnsiv mchnisms 9. hs numr of vntgs s potntil ntioxint: it ffctivly nutrlizs O in living clls, n, unlik most known ntioxints, which r unl to succssfully trgt orgnlls 3,iths fvorl istriution chrctristics: it cn pntrt iommrns n iffus into th cytosol, mitochonri n nuclus. Dspit th mort ruction ctivity of, its rpi gsous iffusion might mk it highly ffctiv for rucing cytotoxic ricls. Its ility to protct nuclr DN n mitochonri suggsts tht it coul ruc th risk of lif stylrlt isss n cncr. mrkly crs oxitiv strss n supprss rin injury cus y ischmi n rprfusion. Inhltion of gs ws mor fficcious thn trtmnt currntly pprov for crrl infrction n, furthrmor, mitigt hptic injury cus y ischmi n rprfusion (K. Fuku, S.., M.I., Y. Ymmoto, I.O. n S.O., unpulish t). This fining inicts tht th nficil ffcts of r not spcific to crrl injury ut cn us for injuris in othr orgns. This stuy suggsts tht protcts clls n tissus ginst strong oxitiv strss y scvnging O. owvr, it rmins possil tht lso protcts from strss y irctly or inirctly rucing othr strong oxint spcis in living clls. For instnc, my inuc cytoprotctiv fctors; howvr, w foun no -inuc chng in th xprssion of svrl gns involv in cytoprotction or ruction (K.N., M.I., I.O. n S.O., unpulish t). Furthr stuis will rvl th mchnisms y which protcts clls n tissus ginst oxitiv strss. cut oxitiv strss my cus y svrl fctors, incluing inflmmtion, intns xrcis, cric infrction, csstion of loo flow n orgn trnsplnttion. For trtmnt, issolv in slin coul sily livr intrvsculrly. For prvntion, sturt in wtr coul ministr. Inhltion of hs lry n us in th prvntion of comprssion sicknss in ivrs n hs shown 69 VOLUME 13 [ NUMBER 6 [ JUNE 7 NTURE MEDICINE

6 7 Ntur Pulishing Group Infrct volum (mm 3 ) % % (%) Stritum Cortx Totl c Nurologicl scor Tmprtur ( C) Boy wight (g) Dy goo sfty profil 31. Notly, hs no risk of flmmility or xplosion t concntrtion of lss thn 4.7% in ir. W propos tht, on of th most wll-known molculs, coul wily us in micl pplictions s sf n ffctiv ntioxint with miniml si ffcts. METODS yrogn n oxygn msurmnts. W msur molculr hyrogn ( ) n oxygn (ioxygn, O ) issolv in solution y using hyrogn lctro (BLE) n n oxygn lctro (Strthklvin Instrumnts), rspctivly. W trmin hyrogn gs concntrtion y gs chromtogrphy (Trmcs). For msuring lvls in loo, w prtrt rts with hprin to voi loo clotting, collct rtril n vnous loo (5 ml ch) in tst tus, n thn immitly injct th loo smpls into clos luminum gs contining 3 ml of ir. ftr complt trnsfr of th gs from th loo to th ir in th clos g, w sujct ml of th ir to gs chromtogrphy using stnr gs, in orr to quntify th mount of. yrogn trtmnt of cultur clls. Ovr -h prio, w issolv yon sturtion lvls into DMEM mium unr.4 MP prssur. W issolv O into scon mium y uling O gs t th sturt lvl (4.5 mg/l), n CO into thir mium y uling CO gs. ll thr mi wr mintin t tmosphric prssur. W thn comin th thr mi ( mium:o mium:co mium) in th proportion 75%:%:5% (vol/vol/vol) n ftl ovin srum (FBS) to chiv finl concntrtion of 1%. For cultur, w put th comin mium into cultur flsk n immitly xmin or O concntrtion with n or O lctro. Thn w fill th cultur flsk with mix gs consisting of 75%,%O n 5% CO (vol/vol/vol) n cultur clls in th clos cultur flsk. W prpr gss mium lcking y stirring th mium, which h n sturt with, in n opn vssl for 4 h, n chck th concntrtion of with hyrogn lctro. In th xprimnts on th os pnnc of (rsults shown in Fig. f), w ilut th comin mium with fourth mium contining 1% FBS quilirt with ir contining 5% CO,inorr Dy Dy f g Dy 3 % % Dy 7 % % No. of positiv clls pr FOV,4 1,6 8 (%) Dy 3 Dy 7 Figur 6 Inhltion of gs improv rin injury ftr 1 wk. Rts inhl % of hyrogn gs uring th 1-min ischmi n rprfusion procur n wr mintin for 1, 3 or 7. () On wk ftr MC occlusion, th rins wr slic n stin with hmtoxylin n osin. Thr rprsnttiv slics r shown. Scl r, 5 mm. () Infrct volums (light-pink rgions in ) wr clcult (n ¼ 6). P o.1. (c) Nurologicl scors wr gr on scl of to 5, s scri prviously 7 : scor, no nurologicl ficit; 1, filur to fully xtn th right forpw;, circling to th right; 3, flling to th right; 4, unl to wlk spontnously; n 5,. Whn rt s nurologicl scor ws jug to twn 1 n, n 3, or 3 n 4, th scor ws st t 1.5,.5 n 3.5, rspctivly. Clos n opn circls rprsnt trtmnt with or without (n ¼ 6). P o.1. (,) Boy wights n tmprtur wr monitor with (clos circls) or without (opn circls) inhltion of % hyrogn gs (n ¼ 6). P o.5, P o.1, P o.1. (f) On ys 3 or 7 ftr MC occlusion, coronl 6-mm sctions from th ischmic cor r in th tmporl cortx wr stin with ntioy to I1 ( microglil mrkr). Scl r, mm (1 mm in th inst). (g) Clls positiv for th I1 ntioy 8, pr fil of viw (FOV), wr count in th ischmic cor r, s inict in f (n ¼ 6). P o.5. Dt rprsnt mn ± s.. to otin th sir concntrtion of ; w thn fill th cultur flsks with th mix gs ilut with ir contining 5% CO. Inuction of oxitiv strss y ntimycin n mnion. W mintin PC1 clls t 37 1C in DMEM mium contining 1% FBS with or without.6 mm in clos flsk fill with mix gss s scri ov. W trt th clls with mnion or ntimycin, which inhiit complx I or complx III, rspctivly, of th mitochonril lctron trnsport chin, n thus prouc O (y cclrting th lkg of lctrons). ftr xposur to ntimycin for 4 h, w ssss cll survivl y mnully counting th clls oul-stin with 1 mm propiium ioi ( clls ll pink) n 5 mm ochst 3334 ( n living clls ll lu) unr fluorscnt microscop. To xmin th protctiv ffct y on mitochonri, w prtrt clls with 4.5 g/l -oxy-d-glucos (n inhiitor of glycolysis) n 1 mm pyruvt ( sustrt of oxitiv phosphoryltion) for 3 min, xpos thm to ntimycin with or without.6 mm n thn quntifi cllulr TP lvls using cllulr TP msurmnt kit (TOYO B-Nt.). Crrl infrction mol. niml protocols wr pprov y th niml Cr n Us Committ of Nippon Micl School. W nsthtiz ml Sprgu-Dwly rts (oy wight: 3 g) with hlothn (4% for inuction, 1% for mintnnc) in mixtur of nitrous oxi n oxygn (7%:3%, vol/vol). W mintin tmprtur (37.5 ±.5 1C) using thrmostticlly controll hting lnkt connct to thrmomtr pro in th rctum, n, t th sm tim, monitor physiologicl prmtrs (using cnnul in th til rtry), incluing loo gss (pco n po ), p, glucos lvl n loo prssur. W ttmpt to mintin constnt lvls of p n po y rgulting th mount of hlothn n th N O:O rtio. W prouc focl ischmi y prforming intrluminl occlusion of th lft mil crrl rtry (MC), using nylon monofilmnt with roun tip n istl silicon rur cylinr s prviously scri 3. Th rts unrwnt MC occlusion for 9 min n thn rprfusion for 3 min; thy inhl gs uring th ntir procss xcpt in th xprimnts corrsponing to Figur 5,. W trt rts with rvon n FK56 using th most ffctiv concntrtions (rfs., 3 n Fig. 5c). ftr th rts rcovr from nsthsi, thy wr mintin t 3 1C. NTURE MEDICINE VOLUME 13 [ NUMBER 6 [ JUNE 7 693

7 7 Ntur Pulishing Group t 4 h ftr MC occlusion, w rmov rins unr nsthsi n slic thm into six coronl squntil sctions ( mm thick). W stin th sctions with,3,5-triphnylttrzolium chlori (TTC) (3%), n thn msur infrct n noninfrct rs using n opticl issctor img nlysis systm (Mc SCOPE, Mitsuy Shoji). W outlin th orr twn infrct n noninfrct tissus, n otin th r of infrction y sutrcting th nonlsion r of th ipsiltrl hmisphr from tht of th contrltrl si. W clcult th volum of infrction s infrct r thicknss. t 1 h, 3 or 7 ftr MC occlusion, w quickly rmov rins unr nsthsi, n fix thm with 1% formlin. W slic prffin-m rins into sris of 6-mm sctions, n stin sctions with hmtoxylin n osin (&E). W thn quntifi th pink rs with grphic nlyzr systm (Mc Scop). For immunostining, w stin th sctions with ntiois y using VECTSTIN BC rgnts ccoring to th supplir s instructions. Sttisticl nlysis. W us SttViw softwr (SS Institut) for th sttisticl nlyss. For singl comprisons, w prform n unpir two-til Stunt s t-tst; for multipl comprisons, w us n nlysis of vrinc (NOV) follow y Fishr s xct tst. W prform xprimnts for quntifiction in lin fshion. Not: Supplmntry informtion is vill on th Ntur Micin wsit. CKNOWLEDGMENTS This work ws support y grnts to S.O. from th Ministry of lth, Lor n Wlfr (17-Chouju-9, longvity scinc; n 17-1, nrvous n mntl isorrs) n th Ministry of Euction, Cultur, Sports, Scinc n Tchnology (16397). UTOR CONTRIBUTIONS S.O. conciv th xprimnts. S.O., I.O., K.K. n Y.K. sign th xprimnts. I.O., S.. n S.O. prform t nlysis. I.O., M.I., K.T., M.W., K.N, K.Y., S.. n S.O. prform th xprimnts. S.O. n I.O. wrot th ppr. COMPETING INTERESTS STTEMENT Th uthors clr no compting finncil intrsts. Pulish onlin t Rprints n prmissions informtion is vill onlin t rprintsnprmissions 1. Wllc, D.C. mitochonril prigm of mtolic n gnrtiv isss, ging, n cncr: wn for volutionry micin. nnu. Rv. Gnt. 39, ().. Ry, P.. myloi prcursor protin-mit fr ricls n oxitiv mg: implictions for th vlopmnt n progrssion of lzhimr s iss. J. Nurochm. 96, 113(6). 3. Oht, S. multi-functionl orgnll mitochonrion is involv in cll th, prolifrtion n iss. Curr. M. Chm. 1, (3). 4. Wright, E., Jr., Scism-Bcon, J.L. & Glss, L.C. Oxitiv strss in typ its: th rol of fsting n postprnil glycmi. Int. J. Clin. Prct. 6, (6). 5. Wintrourn, C.C. Biologicl rctivity n iomrkrs of th nutrophil oxint, hypochlorous ci. Toxicology 181, 37 (). 6. Chinopoulos, C. & m-vizi, V. Clcium, mitochonri n oxitiv strss in nuronl pthology. Novl spcts of n nuring thm. FEBS J. 73, (6). 7. Sur,., Wrtnrg, M. & schlr, J. Rctiv oxygn spcis s intrcllulr mssngrs uring cll growth n iffrntition. Cll. Physiol. Biochm. 11, (1). 8. Turrns, J.F. Mitochonril formtion of rctiv oxygn spcis. J. Physiol. (Lon.) 55, (3). 9. Shu, S.S., Nuuri, D. & nrs, M.W. Trgting ntioxints to mitochonri: nw thrputic irction. Biochim. Biophys. ct 176, 665 (6). 1. Liu,., Colvitti, R., Rovir, I.I. & Finkl, T. Rox-pnnt trnscriptionl rgultion. Circ. Rs. 97, (). 11. Mur, F. Discovry of som of th iologicl ffcts of nitric oxi n its rol in cll signling. Biosci. Rp. 4, (4). 1. Buxton, G.V., Grnstock, C.L., lmn, W.P. & Ross,.B. Criticl rviw of rt constnts for rctions of hyrt lctrons, hyrogn toms n hyroxyl ricls ( O/ O - ) in quous solution. J. Phys. Chm. Rf. Dt 17, (1988). 13. Ohsw, I., Nishimki, K., Ysu, C., Kmino, K. & Oht, S. Dficincy in mitochonril lhy hyrogns incrss vulnrility to oxitiv strss in PC1 clls. J. Nurochm. 84, (3). 14. Stsukini, K., Urno, Y., Kkinum, K., Mjim,.J. & Ngno, T. Dvlopmnt of novl fluorscnc pros tht cn rlily tct rctiv oxygn spcis n istinguish spcific spcis. J. Biol. Chm. 78, (3). 15. Tomizw, S. t l. Th tction n quntifiction of highly rctiv oxygn spcis using th novl PF fluorscnc pro in rt mol of focl crrl ischmi. Nurosci. Rs. 53, (). 16. Kmiy,. Mutgnicitis of 8-hyroxygunin n -hyroxynin prouc y rctiv oxygn spcis. Biol. Phrm. Bull. 7, (4). 17. Ptrsn, D.R. & Doorn, J.. Rctions of 4-hyroxynonnl with protins n cllulr trgts. Fr Ric. Biol. M. 37, (4). 18. Flick,.M., Mhn, B.. & Myrs, R.J. Prmgntic rsonnc spctrum of th 1 D g oxygn molcul. J. Chm. Phys. 4, (1965). 19. soh, S. t l. Protction ginst ischmic rin injury y protin thrputics. Proc. Ntl. c. Sci. US 99, ().. lstrp,.p. Clcium, mitochonri n rprfusion injury: por wy to i. Biochm. Soc. Trns. 34, 337 (6). 1. Lipton, P. Ischmic cll th in rin nurons. Physiol. Rv. 79, (1999).. Frrri, R. t l. Oxitiv strss uring myocril ischmi n hrt filur. Curr. Phrm. Ds. 1, (4). 3. Nito, C., Kmiy, T., U, M., rii, T. & Ktym, Y. Mil hypothrmi nhncs th nuroprotctiv ffcts of FK56 n xpns its thrputic winow following trnsint focl ischmi in rts. Brin Rs. 18, (4). 4. Tk, J. t l. novirus-mit gn trnsfr to ischmic rin is ugmnt in g rts. Exp. Grontol. 38, 4349 (3).. Zhng, N. t l. Ervon rucs rly ccumultion of oxitiv proucts n squntil inflmmtory rsponss ftr trnsint focl ischmi in mic rin. Strok 36, (). 6. Lich, L.. & Grott, J.C. Clinicl trils for cytoprotction in strok. NuroRx 1, 467 (4). 7. Murkmi, K. t l. Mitochonril suscptiility to oxitiv strss xcrts crrl infrction tht follows prmnnt focl crrl ischmi in mutnt mic with mngns suproxi ismuts ficincy. J. Nurosci. 18, 13 (1998). 8. Ito, D. t l. Microgli-spcific loclistion of novl clcium ining protin, I1. Brin Rs. Mol. Brin Rs. 57, 19 (1998). 9. Bjlkovic, G., Nikolov, D., Gluu, L.L., Simontti, R.G. & Gluu, C. Mortlity in rnomiz trils of ntioxint supplmnts for primry n sconry prvntion: systmtic rviw n mt-nlysis. J. m. M. ssoc. 97, (7). 3. Jms,.M., Cochm,.M. & Murphy, M.P. Mitochonri-trgt rox pros s tools in th stuy of oxitiv mg n ging. Mch. ging Dv. 16, (). 31. Fontnri, P. t l. Chngs in mximl prformnc of inspirtory n skltl muscls uring n ftr th 7.1-MP yr 1 rcor humn iv. Eur. J. ppl. Physiol. 81, 338 (). 694 VOLUME 13 [ NUMBER 6 [ JUNE 7 NTURE MEDICINE