Rea卜 time rrlonitoring ofliver damage during experimental
ischaernia― reperfusion using a nitric oxide sensor
Ao Nakagawal,I Yokoyalnal,Ho Suzuki5,K shoji3,Yo Watanabe4,A.Imamura2,To Kokuryol
and ⅣIo Naglnol
D市 iJOn OfSur」 c■ 1 01lCOloP,Department of Surgc7,Nagoya Ulllversi"CTraduate School ofMcdicinc,and IIntcr Mcdical Company Li面

ted,

Nagoyボ FatJ● of Human tttsand Sdence,U」 vcrdけ ofHuman tttsand Sdence,■ Dcparunent OfPharmacO1087,Hi3h TCChndott Research
Ccntre,ShOwa Pharmaceuical lTlliversiり ,Toヽ 7o,Japan,and'Depart■ ■cllt OfLife and RcproductiOn Sciencc,Scction of BiOchcnistり ,Universiリ

OfⅥ rOna,I‐ erona,ItaけCb′ フ ο″rFr″ び´ゎ:Dr Y LkOyalna,D市 isiOn OfSurJcJ OnCO10gy,Deparuncnt of Surge7,NagOya Un市
̀ψ
65 Tsurumai cho,ShOwa‑lm,Nagova 466‑8550,Japan(e̲mail:)γ okO@lllcd.nagOya― u acip)

ersiり Graduatc School ofMcdた inc,

Background:Hepatic ischaema― repel■ lsion cD iniury mり lead to liver damage duHng li■ 7er surgew,
and intrahcPadc nitHC O対 de輻 0)lCVClS mγ Play a rcDle in d■ is context.The aim Of ths stll"waS tO
demonstrate real― time changes in intrahepatic NC)concentration duHng IR and tO correlate pOtential
hepadc NO production、 ath li■ 7er damage using a selectlve NO sensor.
Methods:Wistar rats were exPOSed to 15 111in of hcPatic ischacnlla fonowed by repettsion,a■er whichchangcs in intrahepadc N0 1cvcls were ineasurcd using an NO sensor.Additionallン ЪratS Werc exPosedto■ ve successive pe五 ods ofIRt each COnsisting of 15 n■ in ischaenlia followed by 5 or 15 n」 n repeJttsion,and hcPadc damage■ 唸s emluated by blood tests and histological examinadOn.Hepadc expresslon Ofttkt,

phospho● 71ated ttkt,endotl■ elial nitHc o対 de sy■ ■thasc(CNOS)and PhOSpholブ ated eNOS was examinedat diferent time points du五 ng and an℃ r IR by westem blot and illlmunOhistOchelllical analysis.

Results:1)u五 ng ischacnua,htrahcPatic NO levels increased and reached a plateau at appro対

lllately

10 min.Repcated 15 min ischacn■ ia‑5 11un rcPc血 sion、、les reduced the mattmum amount of NO

produced du血 g ischacmia graduall17,and almost no NO production was observed duHng tlle n■ll period

Of ischaenua.卜 J(D production folloM"ng repcated ischaclnua was proportlonal to the degree of hepatic宙abniけ .PhoSphoり lated cNOS was upregulated and correlated witllthe leve1 0fNO pЮ ducdon durlng

hepadc ischaenua.
Conclusion: IntrahcPatic N(D levels decrcase during rcpeated IR in mts. Real―
intrahepadc NO levels is useil for tlle predicdon Of IR―

time mollitoHng of
epeHmental hК r

related liver il■ iuヮ durlllg

surgerア・

Surgical relevanceusing a selectlve NO sensor.ARer repeated episodes of 15 nlin
ischaclllia and subsequent rcPerftlsiOn,the rnaxlmlllln amount of

HcPadc iscllacmia― repensioll(ID in,Ш り may hduce va五 able

NO prOduced dwing ischacmia gradually decreased.Tl■

degrees Of postoperatlve liver damage because tolerance to IR

ofNTO produced f0110、 mg repeated ischaenlia、 verc PrOporuonalva五 es bewcen padents.¶ herefOre,a de宙 ce dlat ctt predic hverability.
tO the degrec ofhepadc、 ■dalllage du五 ng hepadc IR wOuld be dcsinble Ln ordertO PCfOrm
safe lNer surgery.

This stu"exP10red tlle real― dme dynamics of hrahepatic
五tric∝ idc(NO)levelS durhg repeatcd hcPatic IR in rats

e levels

Rcal― time monitoring ofintahcpatlc NT0 1cvels using sclcavc

NO sensors is useil fOr the predicdon ofIR―

related Lver inluW

and mγ beemplwed h the itureasat∞ lto prevent L

rtturydu五 ng liver sur.・ c理 ァ.

Paper accepted 12 Apri1 2012

Published online 24 M等 2012 in ll‐llcy Onhne Libra,(、 、、町 b卜 .CO・ ukl DOL 10.1002/bis.8817

lntroductlon
Nit五 cO対 de cヽ 0)iS a key protecdve molccule that plays

citrullinc to prOduce N(D.T、 vo isozymes of NOS,cndo―

thelial(cNOS)and induciblc(lNOS)NOS,play an impor―a crucial role in lllaintaining the hepatic inicrocirculatiOn.

tant pathopttd010」 cal r01C in thc hver.The rclativeけ

Nit五 cO対 dc synthasc(NOS)metab01iZes L― arginine tosman(piCOmolar tO nanolnola⇒ quanddes OfNO produced

0 2012 BridshJOulllal ofSurge■ :SOcieり Ltd

Published byJohn｀1lley&Sons Ltd

32ザ r71・ /7.ル″′
7,′ ′ S″ ag′ y2012,99:H20‑■ 28ゲLiver damage during experimental ischaenlia―

1121

reperfusion

depcndcnt actlvation ofcNOS,

constitudvely expresscd in sinusOidal cndOthelial cells,

was hyTOthcsizcd that hcpadc NO product10n is associatcd
with livcr宙 abiliw and that mOnitOring ofintrahepatic NO

are ilnportant in inaintaining thc hcpatic inicrocirculatiOnlevels cOuld bc uscd tO prcdictlivcr damagc during repeatcd

undcr steady― state cOnditionsl(Fig.ゴ ).In cOntrast,during

IR strcss.

by thc calrl10dulin/calcium―acutc ischaenlia the livcr upregulates iNOS and prOduces

largc(micrOm。 lar)quantitics of N02.upregtlladOn Of
ilゞ

Methods

OS is probablyncccssav to COpc、 vith thc suddcn hcpaticischaen■ ic stress,but tirnc(hOurS)iS rCquircd to upregulate

gcne exprcssion and prOtcin synthcsis3 4.Prcvious reports

havc clarincd thc mcchanisrll of callnOdulin/calcium―
indepcndcnt rcgulation Of NO production by phospho―
Aよ t― dependent e卜JOS phosphottzlatiOn undcr ischaenliccOnditiOns5 8(F431).Vla thiS mcchanism,largc quandtiesof N()lnay bc prOduccd in sccOnds,bccause this prOccss
is regulatcd by phOspho巧 ′ladOn Of assOciated prOtcins anddOcs nOt rcquirc uprcgulation Ofgcnc cxprcssion Or protein
synthesis.

Al■ ilnals

A totn1 0f 162 111alc

ヽ
Aristar rats(250‑300g, 8、 「ccksOld; SLC JAPAN, 卜」agova,Japall)wcrc hOuscd in a
tcmpcratt11lc―

and hulnidih′ ―cOntrollc(l cnvirOnlllcnt on

a 12‑h light― dark cyclc and aHO、 vcd access to food・crc、
v:ltcr ′a ll(l stcrilizecI 、
7″ //み ″″ . 測 l CXperinncnts
77夕

approvcd l)y thclヽ agoyそ 、L丁 1livcrsi、「C01nnlittcc 01l Allinlal

Rcsearch, ■1ld aH cxperilnental aninlals rcccivcd hunlane
carc in accorclancc、 vith thc National lnstitutcs of Hcalth

・

′
η′
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ルカ r/7′ G7′ ′′″ し1ヤ げL″ 力θ
ッH″ //7ノ ″た。
Publicatioll,G′ ′7・Intrahcpatic卜 」
O lcvels inay changc f0110wing hcpadc
IR9, but thc rcal― tilllc dynanlics arc still unclcar. In

PartiCular,the dynanlics ofintrahcpatic NO immcdiatcly
aftcr inidation of ischacnlia havc ncvcr bcen evaluatcd
Rcccntly,sclccdvc N()scnsors、 vcrc dcvc10pcd to lncasurc
卜」
0 1cvcls both ′
″υ′
72υ ′
ォ
フ
っ1113, and havcυο10 12 and グ

̀′Speciflc llltHc O対 de sensor
lo― electrOdc potcntiOstatic L〜
Thc t、 、(pr01)C dianlctcr 200

IN‑11l NO scllsorμnl)was purch:lsccl llolll lntcrAlc(lical(NagOyn,Japall).ThiS NO scnsor(F4g SI,Suppordllg

becn uscd tO mOnitor rcal― tiinc NO productiOn in

inお rlnati011)can dctcct NO collcclltrations as low as lbrain10, hcartll and kldncy12. Howcver, ノηυノυθstudics
dcmOnstradng thc rcal― timc dynanlics ofintrahcpatic NO

ヽlS p01arizctl tllld calil)ratcci to conirin its tlccurac)7.

nn101/1 BcflDrc and altcr an cxPcl‐ ilncllts, thc N()scllsOr=を

lcvcls during rcpcatcd IR havc nOt bccn pcrfOrmcd.

Thc purposc of this stlldy 、
vas tO characterizc rcal―
tilne changes in intrahcpadc NO levels during repeatcd
hcpatlc IR using a sensitive and sclcctivc卜 T()scnsor in
rat l市 cr.As lヾ O is prOtcct市 c during ischacmic stress,it

Rats u.erc anacsthctizcd、Fith PCntol)arbital sOdiunl(15 nlg/kg illtrapcritoncal iniCCtiOn)oll ll hCnting pnd
Alter uppcr abdolllillal tr21ls、 ァ
crSC l,ParOtOnl)T,a worに ng
clcctl‐

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♂７Acute ischaemia
CaM/Ca― independenl

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Normal oondlion
CaM/Ca‐ dependent

Surgical procedures and expeHIll■ ental protocol

vas illscrtcd 5 1〕 1111 (lCCp illto thc lclt 101)c
odc 、

vas attachc(lOf thc livcr an(l a rcfcrcncc clcctrOdc 、tO thc surfacc Of thc livcr. Thcrcattcr, thc 、=or鳳 1lg
clcctro(lc was a110wcd tO stabilizc lclr 30 min ulltil thc

rccording lillc was balanccd.This lcvcl was sct as zerovas
ancl the rclative changc in intrahcPatic N()lcvcls 、
nlcasurcd ill subscqucnt cxpcrilnents To stop hcptltic「itll
inf10w, thc hepatOduO(lcllal liganlcnt was clanlpcd 、
a vasctllar clip(BEAR ⅣIcdic COrporation,CI〕 iba,Japall)and rccirculatiOn、 ras cstablishcd by rclcasillg thc、 ・SCular:、

clip Thc rcspOnsc ofthc clcctrOdc tO N()、 =as rccordcd asclcctrical currcnt.Blood alld livcr sanlplcs、 vcrc hanrcstccl
at dilfcrent tinle poillts during IR.In a scparatc cxpcrinlellt,
rcpctltcd clcles Of 15 nlin Of ischaenli:、

ltlHo、 cd

1 5 111111 0f rcpcrfusion、 'crc applicd ill solnc rats(″

Fig.l Nitric o対de(NO)productiOn in dlc nOrmal condidOn andby 5 or=12)

Sample preparationin acutc ischaemia CaA/1,calmOdulu■ ;Ca,calcium;P,phosphate;
11ヾ OS,induciblc nitric o対 dc s)ndlasc;c卜 」
OS,cndOthclial nitric

Tissuc stinlples tOr、 =cstcrn l)lot anal)rsis and clヾ ()S acti、O対 dc synthasc

assays、 vcrc obtaillcd altcr O,1,3,5,7,10,1 5 and 30 11lill of( 2012 Bridsll Jourila1 0f Surge■ i Sociew Ltd
Pt]blisllcd b)JO111■ Vilc)&SOils Ltd

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A Nakagawa,Y.Yokoyama,H.Suzuki,K.Sho,i,Y Watanabe,A.lmamura,T.Kokuryo and M.Nagino

1122

tOtalischaemia.Samples were also Obtaincd after 15 n■

in Of

and
10 mmo1/1 sodium nuoride(Cell Signaling Tcchn01089.

CalbiOchcm Mcrck,San Dicgo,California,US2りヽchacmia f01lowcdけ 5 min Ofreperh」 on.Each samplc

was collected frOm One rat,which was then kllcd.Tissuc
samplcs wcrc cut inllncdiatcly intO S― mm cubic sections
and kcpt at‑80° C until use.Six to tcn rats were uscd forcach grOup and dmc pOint.

Tissuc samplcs were hOmOgcnizcd using Bcad Smash
120 い「akenyaku, Kyoto,Japan),Centriigcd at 10 000 g
at 4° C for 10 min,and the supernatant was collected.

ヽヽ1lile thc supernatant、vas bcing sdrred, thc NO

scnsor was dippcd intO thc supcrnatant. Subscqucntly,

l mm01/1 na宙 n̲adcninc dinucleOtidc(Ccll Signaling
Tcchnolo8o,10 mmo1/1 tetrahydrObiOpterin(CalbiOchcm

Administration ofnit五 co対 de synthase inhibitor
To inhibit NOS acti宙 り,the nOn― spcci6c NOS inhibitor
ArG̲nitro― L― arginine methyl estcr(L― NAⅣ IE;Ccll Signal―

MCrCD and lo mm01/1 rcduced nicotinamide― adcninc

ing Tcchn010〔 y,Danvcrs,Massachusctts,USの ,WhiCh

This solutiOn was madc with, Or withOut 10 μmo1/1
calmodulin(Ccll Signaling Tcchn010"and l mmo1/1

inhЫ ts bOth iNOS and cNOS,15 mg/峰
tonealサ

intraperi―r thc
60 min befOre hepadc ischacmia14, 。

sclcctive inhibitOr Of iNOS N6̲(1̲iminOcthメ)― L― lysinc(L― NIL;Ccn Signaling Tcchno10gめ , 3mノ kg intra̲
venousサ 15 min bcfOrc hcpadc ischaemia f0110wed by an

dinuclcoddc phosphate(CalbiOChem ⅣIcrckl were addcd.

calcium (Sigma, st LOuis, ⅣIissouri, USAl.Finally,
l mm01/1L― argininc(Substratc Of NOS;Cell Signaling
Tcchn010gy)was addCd and changcs in NO lcvcls wcrc
mcasurcd.

inisiOn Of l mg pcr kg pcr h15,waS inicctCd in separate
animals.
Iη J詢%visualizatiOn of nit五Iπ υ″
辞、 endothelial

nitric o対de synthase activity

To宙 sualize NO,4,5‑diamin01uOrcsccin diacctate(DAF2‑

assay
̲411

co対 deDA; Sigma)staining was pcrformcd on 5‑μ m thickノηυ′
r7っ cxpcrilllcnts、 vcrc pcrformcd in a box ttncd

with nitrOgcn gas at 32° C bccause NO is unstablc
vas
in the prescnce of Oxygen. Each liver samplc 、

sOakcd in a c01umn、
vith l lnl phOsphatc buffcrcd

salinc(PBS)COntaining l mmo1/1 phcnylmcthylsulphOnyl■Ozcn hcpadc tissuc sccdons(bCfOre and aftcr 15 1nin
ischaemia).ThC ScctiOns wcrc incubated in 10 5 mo1/1

DAF2‑DA diluted in PBS buffcr fOr 2 h.Samplcs wcrc
then washcd Out thrcc tlmcs using PBS and mOuntcd.
DAF2‑DA staining was cxamincd under luOrcsccnce on

luOride(PMSFl,l mo1/1 EDTA― frec complctc proteasean Olympus nuorescence microscopc(IX71;Olympus,om

TOけ 0,Japan)CqdPpCd価 th an cxdtadon(447 517 nm)

inhibitOr, l mmo1/1 sOdium Orthovanadate(all fl・合ヽｂ Ｅ ュ︶ ｃＯ ´Ｅ ´ｃｏｏｃＯＯ Ｏ Ｚ

今ヽ
ｇ だ ００ｃｏｏ Ｏ Ｚ
ち Ｅ こ ｃ２ ¨

0

5

10

15

20

25

30

35

40

‑5L

45

0

Time(mln)

a NO leveldunnglRb Ettect of NOS inhiblors

Fig.2a■ ■trahcpatic nitric o対 dc(NO)level during ischacmia― rcpcrisiOn(IRl in thC ratl市 cr using a sclccivc NO scnsor(7'=8).
b Effects Ofthe nOn― specinc nitric。 対dcspd■ asc NOS)inhibitor(NC― nltro― L― arghu■ e me■ yl ester,L―S｀ E)or spCCilC ulduciЫ e

NOS ulhibitOr w6̲(1̲imll10ct,1)― L― レsinC,L― blIL)on intrahcPadc NO levcls durll■ g IR(72=3 pcr group).Valucs arc mean(sCm).

Ncgat市 c valucs fOr lヾ 0 0bscwed during rcpcrision hdicatc that NO lcvcls wcrc 10wcr than at dlc start ofischaemia◎2012 BridshJournal ofSurgew Socicw Ltd
Pubhshcd byJohn llriley&Sons Ltd

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1123

Liver damage during experimenta:ischaemia― reperfusionand emissiOn(496‑576 nm)green lltcr fOr DAF‑2
luOrcsccncc16.

Westem blot analysis

Tissuc samples were m破 ed with Laemmli samplc
burer contai面 ng l mmo1/1 PⅣ ISF,l mmo1/1EDTA― freecomplete protease inhibitor,1■ 1lno1/1 sodium orthovana―auid was c。1lcctcd as a lysatc and clectrOphoresed On
sodium dOdccyl sulphate― polyacrylamide gels,transfcrrcd
tO p01y宙 nylidene diluo五 de membrane(lmmObi10n,IIlil―lipOre, BillcHca,ル Iassachusctts,USA)and then prObcd

with andbOdies,signals、vere detected with an enhanced
chcmiluminescence systcm(Nakalai,Kyoto,Japan).

■ssue samples were tted immediateサ

in neutraln,and then either

buffcred fOrmalin,embedded in Para

staincd Mth hacmatOxylin and cOsin or subjcctcd tO
eN(DS, andiN(DS using the automated slide preparation systcm
inlnunohistOchenlisty for eNOS, phospho―

Discovcry XT α cntana Mcdical Systcms, Tucson,
ArizOna,US2り .BefOre staining,parahcated at 65° C fbr 15 min in a para

‑1

1

3

4

5

6

Time(min)b 30 min ischaemlan scctiOns weren ovcn and blockedwith nOn― fat inilk.The staining proccdurc was carricd out

2合ヽ
ち Ｅ ュ︶ｂ＞聖 Ｏ Ｚ ｃ一ｏｏＣ● ０

Histological evaluadon

ぞ ｂ Ｅ ュゴ ｏ＞⊇ Ｏ ｚ

date and 10 1nmOν l sOdium ttuoHde,and then homOge―
nizcd and ccntHfuged as describcd abOvc.The supcrnatanta 3min ischaemia

0

1

3

7

15

30

1schaemia lme(min)C

わ 無 seNOS assay

accordingtO thc manufacturcr'sprotoco1 0″ entana Mcdical
SyNstems).The and― cNOS antibody(BD Biosdcnce,San
Diego,CalifOmia,US→ ,and― phOspho― eNOS andbody(CCll Signaling Techn010gyp and and― iNOS andbo"(BD BiOsciencc)were diluted in Discovery° Ab Diluent
ycntana Mcdical Systcms).

Biochenical assay ofblood samples
Blood samples were taken fOr measurcmcnt of aspartate
aminOtransfcrasc ぃ ST)and alaninc aminotransferasccALT)け standard laborato7 methods.

Fig.3 RcPresentadveブ″υ′け、data shO、 vhg changes in nitricaidc(NO)lcvCIS h l市 cr hOmOgcnatcs ofsamples followhg
a31nm and b 30 m菫 1 0fischaenlia durmg ad■ linistradon ofburered saline:1,lavln― adcnhe

COCnη"nes inphosphate―anudcodde
l nlm014;2,tetraけ
drobiOpte五 n10 mmo14;3,
rcduced nicounanlide― adenn■ e dhucleoddc phosphatc
10 mmo1/1;4,dle substratc L― ar.CInhc.c NO producdon hhOmOgcnatcs in thcノ ″υブ″οcndOthclial nitrlc o対 de synthasc(eNOS)acuVlty assγ with Or withOut cahnodulhm。 1/1)/Calcluln(lmm。 1/1)(CaM/Ca)(π =6‑7 in cach
(10 μgroupゝ valueS are mcal■ (s.e.m。 )s10pe at each dme pOint(the differential coettdentl Was
calculatcd to estimatc thc potcndal for NC)productiOn.

Statistical analysis

Results
Data are expressed as mean(s.e.m.).Stadstical differ―■th Studcnt's
cnces be●veen 3vO groups werc compared、 マιtcst.The level of siglllicancc was sct at P<0・ 050.Thc

Ⅳlonitoring Ofintrahepatic llltHc o対 de levels
during ischaeIIlla― reperfusion

integrated value Ofthe NO curvc← he area under thc curvc;

During ischaenlia, intrahepatic

AUC)waS Calculatcd tO csdmatc total NO producdon.Thc

卜」
0 1cvcls graduaHy
increascd and rcachcd a platcau at appro蓋 matcl17

0 2012 BridshJourna1 0fSurgew Sociew Ltd

WヽVヽ アbiS

Published byJohn l■ lley&Sol■ s Ltcl

CO.uk

″
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B″ r7・・/7ヌ ο
9FS″ 電 y2012;99:1120‑1128
̀′A.Nakagawa,Y.Yokoyama,H.Suzuki,K.ShoiL Y.Watanabe,A.lmamura,T.Kokuryo and M.Nagino

1124

Phospho― Akt

■■
―

eNOS
助

山 ∝eNOS艶 ‖77灘

憲 ざ‐

iNOS
Actin
β―

1:}

ischaemia(min)ReperusiOn(min)a western blotting
O min

30 min3 min

Phospho― eNOSb immunohistochemistry
臓g.4 a Expressiol■

Ofttt,PhOsPhoり ated Akt lphOSphO― 測 くo,CndOtl■ cliJ nit五 co対 de wthase(cNOS),phOSphOwiatcd cNOSlphOSPho― cNOS)and induciblc nitHc o対 dc synthasc(iNOS)at difFcrcnt dmc Pohts ofhepadc ischacmia― rePcriSiOn(wcstCm b10o.
Ac・ in was uscd as a cOntr01.blmm■ lnOhistochemistv for cNOS and PhosPhO―
cNOS protcElin d■ cO― ,3‑and 30‑min hcpaticβ―ischacmia groups(orlginal magnincadon× 100,ulset magnincadon× 400)

10 min(Fな 2″ ).ThCre was no hrthcr increase in NO

NO productiOn c43 2ι ),suggCSing that the high NO

conccntradon cvcn aftcr 30 min of ischaemia(data nOt

prOduction duHng ischacnlia was not dependcnt On iNOS

shOwll).On rCpcrhsion,aftcr a small increase,NO lcv―

aCuⅥ W・

cls quickly retllrncd tO bascline.Negadvc valucs for NO
obscrvcd during rcpcrftlsiOn indicatc that NO levcls wcrc
10wer than at thc start Of ischaemia.Following adminis―
tradon of the nOn― spccinc NOS inibitor L― NAVIE,N(D

prOducdOn was almOst cOmpletely inhibitcd(Fな・2多 ),indi―Iη υz計、 assay

IIigh NO producdOn was observed in livcr homOgenatcs
Of samplcs takcn fOHOwing 3 1nin of hepadc ischacnlia

cating that NC)biOsynthesis during IR was dependent On

C4g.′ ′).NO producdOn dccrcased gradually as thc lcngth

NOS.L― NIL(specinc inhibitOr OfiNOS)did nOt inhibit

Of ischaenlia increascd, and ahnOst nO incrcasc in NO

02012 BrijshJOuma10fSurgewSociewLtdやヽフヽV.biS CO uk
ヽPublished byJohn WllCy ttt Sons Ltd

B7・ 7′

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777´ ノ S″ ry,y2012;99:1120‑1128
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1125

Liver damage during experimentai ischaemia― reperfusion

concentradon was obscrvcd a■ er 15 or 30 1nin of hepadc

1evels gradually dccrcascd and wcrc alinost undctcctablc 15

ischaemia cな .′ ι The addition Of calmodulin/caldllm

or 30 min after the initiation of ischacnlia. Phospho―

did nOt alter NO prOducdon, indicating that NO prO―

触 t levcls wcrc rcstorcd aftcr rcpcrilsion but were nOt
hghcr than during the frst pcriod of ischaenlia.「 Fhelcvcl ofphosPhorylated eNOS at Serl177 showed changes
similar to thosc of phosphO― Akt.In contrast to the cNOS)・

ductiOn duHng ischaelnia was not dcpcndcnt on callnod―

ulin/calcium cな。わ 。
Iη ∫″協 宙sualization ofllltnc o対 de

PhOsphoryladon,the expression ofiNOS protcin was weak
and showcd nO changc duHng IR.

NO was visualizcd in endOthclial ccns in pCriportal
area%this was mOrc prominent a■ er 15 min ischaemia
than at baseline● efOre ischaemia)Cな .S2,suppordng―

infOrmatiOn).

eNOS was exprcsscd prcdOminantly in the periportal

Westem blot analysis

arca along the sinusOidal endothelial ccns in the Lver;its exprcssion did nOt changc during IR Ftt.イ の.UndCr

unohistochelmstry

non― ischaenlic conditions phosphO― eN(DS was expressed

PhOsphO― Akt levels, assumed to be upstream of eNOS
phOsphOryladOn,were much higher at l and 3 min a■ er
inidadOn ofischaemia than at baseLne Ftき イ″).HOWevcr,

weakly,but aftcr 3 min of ischacmia expression becamc
much strOngcr in the periportal arca a10ng thc sinusoidal
endothclial cclls.PhOspho― cN(DS was all■ ost undetectablc

Ｃ一
ＯＥ じ ｂ＞２ ０ ｚ

合ヽｏＥ こ ０＞聖 Ｏ ｚ

Time(min)Time(min)a15

min ischaemia‑5 min reperfusion cycles

b15 min ischaemia‑1 5 min reperfusion cycles
600

800

600
合ヽ
∽一
て つ︶トコく

今ｂ 一
ｔ 己 卜∽くlschaemic perlodlschaenlic periodd AL丁 leve:s

C ASTlevels
Fig.5 a Changcsin htrahcPadc nidc o対de(NO)lcvClS du五 ng ischacmia― repcrftlsion h thc rat l市 er by rcPcattg 15 min ofischaemia

and a subsequcnt 5 1nin OfreperisiOn Or b 15 1nm ofischaelnia and a subsequent 15 1nm ofrcpcrftlsiOn c,d Scrlm levels Ofc asPartate

amhOtransferase CiS⊃ and d alanlne aminotransferasc(ALTh aftCr thc third,fourth and l価 pe五 ods ofischacmia.Vallles arcmean(S.e.m.).=Pく

0・

な簿 5 min repcrision(StlldCnt's r tcso
050υ′

◎2012 BriishJouma1 0fSurgev Sociew Ltd
Published byJohn｀ ヤ
lley&SOns Ltd

ヽVヽVヽ V biS.CO・ uk

B″ rrFZル %物 ′′√S″ γ 72012;99:H20‑■ 28『

A.Nakagawa,Y.Yokoyama,H.Suzuki,K.Sholi,Y.Watanabe,A Imamura,T.Kokuryo and M.Naginoa■ er

30 min of ischaemia(ag%).ExPrcsSiOn of iNOS

by immunohistOchemist7 was weak during IR(data nOt

Of intrahcpatic NC)lcvcls during rcpeated hcpatic IR
using a selecdve 卜T(D sensOr. Intrahcpatic 卜」C)levcls

shOwn).

incrcascd gradually durillg ischaeinin and rcached stca(lystatc appro対 lnately 10 nlin a■ cr hcpatoduOdenalliganlcnt

Ⅳlonito五ng ofintrahepatic llltrlc o対 de levels

du五ng repeated ischaenua― repeJhsion

Occludon.01l reperision,aftcr a sm■ 1l increase,NO
lcvcls quickly decreased to around basclinc levcl.「 Fhcsn.an incrcasc of N(D at thc bcgillning of rcpcr負 lsion

Thc peak Of NC)produccd during ischaenlia dccreased ina stcp、 vise inanner by repeadng 15 1nin ischacnlia‑5 ininrcperision cycles o鷲 .力

inay be due to a sudden supply of Oxvgen frOm thc
gastrointestinal circulatiOn,as production oflヾC)requircs

Almost nO increasc in thc lcvcl

thc substrntc L― arginine and owgcn.A notable obscration

ofNO was dctectable during thc lfth period ofischaemia.
Levels did nOt return to baselinc and rcmained at arOund()scnsOr、 vas thc high an10unt oflヾ (),in thc
using the卜 」)・5 μ
mo1/1 cvcn aftcr rcpcrfusion.Howcver,NO production

was signincantly grcatcr with a longer(15 min)than a
shOrter(5 min)reperidOn pe五 od c4g。 ル).Du五 ng cachrcpcrilsion period,NO levcls rcturncd allnost to basclinc.

Or(lcr Ofinicr011lolar concentrations,produced in responsctO ischaclllic strcss.This indicatcs that thc livcr has an

こO

cnornlous potential to prO(luce largc quantities of卜quicHy in rcsponse to ischaelllic strcss.

cNOS is thc NOS isofrDrln rcsP01lSiblc ttr illaintain―

ing systcnlic bloOd prcssure, vascular rcn10dcning andBiocheIIllcal assay ofblood samples and tissue
hist010gy
Serum lcvcls Of AST and ALT were signincantly greater
after 5 min rcpcrisiOn than a■ er 15 min repcrftlsiOnι
,の・TheSe differences wcrc cttn more prominent(Fな 夕
6ha■ er the 6fth period of IR,and hist010gy conirmcd

more sinusoidal cOngcsdon and hcpadc Parcnchylnal
damage in the shOrt reperfusiOn grOup, indicadng thc
prcsence of scК re circulatoヮ failurc(F4,S,Suppordng
infOrmatiOn).

Conceptual diagram ofthe nltHc o対 de curve
analysis
cicnt in eachThc AUC and the peak diffcrcndal coeヽchacmic Pc」 od gradualサ dccrCascd宙 th rcpcated IR.
This dccrcase was more pronlinent fOHOwing 5 111in rcpcr―

angiogcncsis20. lTndcr nornlal phvsiological conditiOns,
rclativcly slnall quantities of N()arc prOduccd throughOS in sillusoidal cndothclial ccns in
the activation of c卜 」
tlle livcr and dif■ lsc a shOrt distance to llcprltic stcnate

ccns, causillg their rclaxation and subscqucllt sillusoidal
dilatatiOn21.Durillg ischacnlic strcss,ilゞ

OS is uprcgulatcd()、 =i(l aill the livcr and produces largc quantities of卜 こ

calmo(lulil1/calcium̲indcpclldcnt mechallisn13.As iNOS
uprcgulatiOn rcquircs scvcral hours,thc illlnlcdiatc changc
in intrahcpatic卜 T()conccntration obsewecl aftcr thc ini―
tiation Of ischacnlia is unlikely to bc cxplaillcd by i》 ヾC)S′がυοstu(ly,a spccinc iNOS
inctiOn.In ict,in thcノ ′inhil)itor(L‑1ヽ IL)did nOt il〕 hibit thc increase in intrahcp―(D lcvcls during ischicnlitl,■ nd、 restcrn blotting an(1atic卜 」

inllnunohistOchcllnist■ = fOr iN()S prOtcill cxprcssion did
nOt shO、 v any increase during ischaclnia.

In additiOn tO thc cOnvcntional lヾ ()lorn■ 1ltiOn throughftlsiOn than a■ er 15 min repcrisiOn(ag.Sイ ,supporting(DS activatiOn, Phos―
callno(lulin/calciunl̲dcpcn(lcnt c卜 」

infOrmatiOn).

phOn/1ntion of e卜 」
OS produces largc tlll10unts of卜 I(DDiscusslon

csizc(l that N(D prOduction llnmcdiatcly atcr ischacnlic
vas incdiatcd by cNOS phosphon/1atiOn, becausc
strcss 、

fcl110wing ccllular stimulati01122.Thcrcお re,it was hypoth―NC)is onc of thc lnOst potcnt vasodilatOrs and is highly

this prOcess does nOt require genc exPrcsSiOn ind Protcill

upregulatcd in response to hcpatic IR strcss ill thc

syllthcsis.In thc present study,PhOsphO―

livcr17.Hcpttic gcncration of卜 JO mav attcnuate sillusoidal

was lnuch highcr l and 3 11lill aftcr starting ischacl■ lia thanperisiOn failurc and ilnprOvc livcr tissuc ovgcnation18 19.

at bascline.HO、 :cvcr,lcveis gradualy dccrctlscd and、(D production during ischaclnin
Thc uprcgulatiOn of卜 」

ahnOst undctcctable altcr 15 or 30 1nin ofischacn■ ia.Thcsc

is prcsunlably a nccessaw step tO COpc、 vith thc sud(lcn

results arc consistcnt vttth thc results Of thcグ

tcrl■ lination

activitv assav and inllnunOhistOchcnlistr丁 .´ rhis hypOthesisof Organ l)lood lo、 v

Thc instabilitv Of lヾ O in aqucous solutions and its highrctlctiviw with othcr m01cculcs makcs its nlcasurcnlcnt
dicult.Consequently,thc klnctics and mcchallisnls thatwas supportcd by the rcsults ofル ′τ
'た'ο

cN()S cxPrcssiOn
rcrc

″υノ
r7り cNOS

studics usillg N()S

inhibitOrs.Thc incrcasc in intrahepatic IゞO levcls during

hepatic ischaelnia、 vas prol)ably duc to the accunlulatioll(leternline 卜T(D levels during hcpatic IR arc still poOrly

Of NO produccd by phospho理

undcrst00d. This studv explorcd thc rcal― tinlc dynan■ ics

̀lated eNOS.The
platcauthat occurrcd apprOxinlatcly 10‑151nin
a■ er initintion●2012 Brids1lJourl101 0fSurge■ ,SOciew Ltd:iley&Solls Lttl
Publislled byJohll｀ へ、、、・bis co uk

θ′
ブ 力、
″″
ノグS″ ,y′ 〕2012:99:H20‑H28
7r7″ ′
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1127

Liver damage during experimenta:ischaemia― reperfusion

Of ischacrnia may bc cxplaincd by the disappcarancc Of

has nOt yct bccn applicd during rcal― tilne hcpatcctOmy inhcpatic ability tO acdvate thc phosphOl■ 71atcd eNOS path―largc aniinals or humans.

way.These rcsults also indicate that thc ccllular capability

to produce NO is liinited whcn ischacnlic dme cxtcnds

DisclosurebcyOnd 10 1■ in.

HepatectomyisachaHengingsurgicalprocedurebccause
Ofthc high risk Of b100d 10ss.TO rcducc b100d 10ss during

hcpatectomy,thc hepatOduodcnal ligamcnt is frcqucntly
clamped(P五 ngle manOcuvre)23, either cOntinuously or

Thc authors dcclarc nO conlict Ofilltcrest

intcrnlittently.In general,an ischaclnia tilnc of 15 inin andReferencesa rcpcrftlsiOn tiinc of 5 inin arc cmploycd.Ho、 vcvcr,thc

l HOric Y,urolf R,Grangcr DN ROlc Or nitric o邁

tolcrance Of the liver to ischacnlic strcss is sdH unclear

and inay vaw in cach padcnt.Thcrefore,thc Optilnal dmc
fOr ischacnlia and rcpcrisiOn shOuld bc dcterlnincd fOr

dcin g■ lt

iscllcnlia― repertusion― induccd hcpatic lllicrovascular/1997;273:G1007‑G1013d),sillcdoll H777、 7P/71・ ブ
ο
2 Yanaglda H,■ ibOri Al,Yoshida H,Habara K,Yninada Al,

individual padcnts,and a dc、 ■cc that prcdicts liver damagc

I【 nlllil,allla Yご

du五 ng hcpatic IR may hclp perform safe livcr surgely.

the susccpdbilitv OfhepatOcytes to conlcr thc illdllctlon Of

Thc peak OfNO prOduction during ischacmia gradualヶdccreased in success市 c15 min ischaemia‑5 min reperi―
siOn γ cles,and alinOst no NO producdOn was detcctablc

け the l■ h periOd Of ischaemia.Neverthdcss,N01ev―
cls、 vere rnaintained around 5 μ
m01/1.NC)producdon was
signincantサ grcatCr with 15 min rcpcrftlsion than v・ lth

5 min rcpcrftlsiOno NOお quicltt scavcngcdけ haCm
m01cculcs in thc b100d.「 FhcrcfOrc, thc NO dynanlics
during reperftlsion may represent the dcgrcc to whichaccumulatcd NC)is 、
vashcd Out by thc recovc7 of thc
hepatlc inacro/111icrocirculat10n.It can be spcculatcd that15 111in of reperttlsiOn inay be sufncient to wash Out accu―

mulated NO ahnost cOmpletely and tO rcstorc rヾ

o levelstO basclinc,cvcn aftcr thc nfth ischacnlic pcriod.

Thc rcpcatcd ischaclllic stress 、vith shOrt intcn/als
induccs a disturbancc Of thc hcpadc nlicrocirculatiOn.
This reduces Oxygen suppサ in thC liVcr and may lcad
to rcduccd adcnOsinc 5′ ―triphosphate generatlon24 andsubscqucnt phosphOrvlatcd cN(DS formation, resultingin N(D pcak during ischaelllia. Incomplctc washout of
CXCCSSiVeケ aCCumulated NO in thel市 cr during repcrftlsiOn
may resultin raised intrahepatic NO levcls cvcn during the

rcpcrision intcrval.This may inalヶthrOugll the prOccss of hpid perO対

damagC thC livcr

dadOn25.ThercfOre,

an adcquate reperilsion inteⅣ al may bc necessary toan。 、v bettcr rccOvc7 of thc macrO/nlicrOcirculadon anddssuc ovgcn lCVCls.Thc rcsults Of this study impサ that
intrahepaticヽ こO measurements can bc uscd to mOnitor
livcr damagc during hepatic surgcw using a sclcctivc N(Dsensor. By hrthcr charactcrizing thc dynanlic cun7cs ofintrahcpatic NO lcvcls using this de宙 cc,it inay bccOmc′′ Hcpatlc ischclllia/rcpcrilsion uprcgュ

1latcs7ノ

induciblc llitric o、 idc s)■ lthasc gcllc cxprcssion.S力ο 2006;
̀た

26:162‑168
3 HurG卜 I,Rw YS,Yun HY,Jcoll BH,Knl Y卜 I,ScokJH″″ノHcpa」 c iscllelllia/reperAlsi。 1l ill rats illduccs iNOS″′
gcnc transcriP」 on by ac」 、=ntion Of lヾ F― kappaB βん
̀/1ごθ70p′ tr R″ Cο 777″ ′″″1999;261,917‑922

4 Kawai K,Yokq'allla Y,k)ku,oT,1ヽ Ъtallabc K,
玉3taga、 va「 F,rヾ agin。 卜l lnchinkOtO,a1l hcrba1 11letlicille,・cヽ clts bcneflcial cflccts lll thc rat li、
cr linder strcss with
hepatic iscl〕 clllin― repcrlllsion antl subscqucnt hcl)atcct()111)「/1″ ″S〃 ′32010:251:692‑700

AIchdi AB,Fishcr AB Sigll■
5ヽ ア
ci Z,劇 ―lillg pathway IOrnitric o五 dc gcllcl・ atlon witll silnlllatcd ischcmia il

ノliャ ″7C力
10w― ndaptedぃ ldOthchal cdls.´ 4″ ′アP/"′ ο、2001;281:H2226‑H2232

̀P/71170ノ

6 Hoshino S,LLnchi Y,Nakajllna M,Klnllra H,Tsuvnina S,
Uelnura K′ ′′′Endodlclinl NO syllthasc(eNOS)
phOsphol■ 71ati。 1l rcg■ 1latcs cOronapF dianlctcr dllrillg
iscllc11lia― rcpcrtusiol)in association widl o通

dativc sti・ csS.

乃で′R′ 7r//̀R″ 2()05;39:481‑489
7 Dimllleler S,Flellling I,Fissld■ aler B,Hcrinann C,Busse R,
Zcillcr MI ActivatiOn Ofllitric Oxi(lc svnthtlsc lll cndOthclial′ 19991399:
cclls byハ よt― dependellt phosph。 ぃFintiOn Ǹ,ノ ″

601‑605
8 Fult()nD,Gratt()1lJP,llrlcCabe TT,FOntall■

J,Fulio Y,

1lЪ Isll K′ ″″′Rcgulat10n OF cnt10thcliunl― dcrivcd nitric

1999;
O」 dc produciOnけ thC protcin ttnasc Akt N7′ /77‐ こ

399:597‑601.
9 LccSH,Culbcrs01l C,Kc)rncszczuk K,Clcmcns AIG.
Dittcrcntial incchanisins Ofhcpatic vascular(1卜

7srcgulatiOn

P/11,ノ οノ、itll lnild τ
ド mOderate ischeinin― rcpcrision′ ′
.ア
‐
̀″
″′
り′
″r L7tで ′P/1ド ノοノ20081294:G1219‑G1226
G/7N′ ′

10 Ynllg Y,Kc― Zhou L,Nillg GAl,ヽVallg卜 IL,Zhcngnpossible to deterlnine an opdmal ischacnlic tinlc andrcpcrftldon intcⅣ althat minlmizc hvcr damagc induccdけ

bralll ischcnlin/reperftlslon in rat hippocalllpus:卜 TO― scnsor

repeated IR.It shOuld be nOted,ho、 vcvcr,that thc prcscnt

incasurcnlcnt and inodcling stlitlv.Ne″

data siinply prO、 ■de a cOnccptual idca and that this prObc

73‑80(0 2012 Britisiぅ

Jourilal of Surge■ =Sociew Ltd

Pubhslled b)JOhil｀ 1:ilcl‐ &Solls Ltd

litritc dllrilng globnl
Dynanlics of nitric O壺 dc and pero、 ■ヽヽヽ biS CO uk

37ブ r7・″′R̀● 2008:33:
′
,び 力
′

″,″ ノr7rS′ ″y7]2012;99:H20‑H28
カル″

A.Nakagawa,Y.Yokoyama,H.Suzuki,K.Sholi,Y.Watanabe,A.:mamura,T.Kokuryo and Mo Nagino

1128

1l MantiOnc KJ,Stcfano GB.A sub nanOmolar real―

dmc nitricO対 dc PrObc:77'υ ′υO nitrlc o対 dc rclcasc in heart Meノ Sσ ノ
M072r″

2004;10:AIIT47‑AIIT49

12 Lc宙 nc DZ,IacO宙 tuル I.Rcal― dmc mcasurement of Ldncy
tllbule nuid nitric O対 de cOncentrations in carヶ diabetCS:

disparatc changes in direrent rodcnt mOdclsヽ

ワ
r7,0∝ iaθ18 Panllen BH,Al― Adili F,Bauerル I,Clemensル lG,Gcigcr KK
Rolc of endOd■ elins and nitrlc de in hcpadc rcpcrisioniniuヮ h the rat.Iセ ′′ gy 1998;27:755‑764"ん

19 Pa対 all M,Kcllcr SA,Bavtta R,Komcszczuk K,Huシ mhq―「
CIcmcnsル lG.ミsctlonallink bc● vccn ETB rcceptors andtain tissue oxygenadon in the normallivcr.
cNOS rna■■
Mic知 ″π″″″ο
722004;11:435‑449

2006;15:87‑92
13 Vlllcncuvc N,BcdiOu F,Voituricz K,AvarO S,Ⅵ laincJPElcctrOchcnlical dctccdOn Ofnit五 c oxldc PrOducdon in7″ ′ωノperfOmnances of● ″。electrOchemicJ sensOrs′ P/7′ ′ミ、プ
ωノMeル ο清 1998;40:95‑100.

14 Morisue A,S、kabayashi G,Shimazuル 1,Tanabe A/1,
A4ukai A4,A4atsumOtO K′ ′′ノThe role Ofnitric o対 de after a
shOrt pcriOd ofl市 cr ischemia― rcpcrAlsiOnノ S′ ′電 R″ 2003;

109:101‑109.
15 1Qdlcl10dacc Ⅳl,Zahmatkcsh M,Sadcghipour HR,

Eslamifar A,TacbJ,ShamS A′ ′″′.Protcll■ uria is rcduccd byhhibidOn Of hduciblc nittic O対 dc synthasc in rat renal
ischcmia― rcpcrAlsiOn uljuv T7′ 77ψ ″″rル η

̀2009;41:

2907‑2909.

16 Haapasalo― Pesu KM,Erk01ahd R,Saarijini S,Aalbcrg V.

Prescrlpdon ofpwchOtrOpic drugs in adolcsccnt Psychiatw

213‑218
17 Albrecht EW,Stcgcman CA,Heerlnga P,Hcnning RH,van

力οノ
2003;199:8‑17」 ′Sessa VVC.Dircct c宙 dcncc fOr thc importance of″′1998;101:731‑736
ノC″″ルυ
21 Rodeberg DA,Chact MS,Bass RC,ArkO■ 71tz A4S,Garcia VF
Nit五 co対 dc:an∝ eniew И″ノS″ 7g1995;170:292‑303.
22 GarcFa― Cardeia G,Fal■ R,Stern DF,LiuJ,SCSSa、 VC.
El■ dothcli」

nit五 cO対 de spthase is rebqllatcdけ

phOspho71adOn and interacts with cavc。

りrOSll■ e777
lin‑1.Jβ あ′Cみ ′1996;271:27237‑27240.
23 Grazi GL,MazziOtd A,J。 宙ne E,Pierangeli F,Ercolani G,
Galucci A′ ′′ス「Fotal vascular cxclusion ofthe l市 cr durhg
hepatic surge■ 7.Selective use,enens市 e usc,or abuscPノ 4,τ み
Srr′

lg 1997;132:1104‑1109.

24ん nscow EK Bralld MD IntcmalrcguladOn OfATP
tllmovcr,glycoケ SiS and o対 dativc phosphOwladOn in rat
hepatocttcS・ E″ ″
οZ′ 7771999;266:737‑749.
ノβ′l・wards in Fhland.ノ Vθ 僣ノユッ
ル″r7D72004;58:

Goor H PrOtecuve role of endOthelial nitric o対

20 Rudic RD,Shcscヶ EG,Macda N,Smithics O,Scgal SS,
cndOthclilllln― derived nitric O対 dc in vascular rcmodelingpcrised Pig cOrOna■ ァarteコ/:comparison ofthedc synthase.

25 Rubbo H,Radi R,Trujillo M,Telleri R,Llyanaraman B,
Barncs S θ′′′卜Titic o対 dc rcguladon OfsuperO対 de andperoゅ mitrite― dependent lipid perO対 datiOn.Format■ on ofnOvci nitrOgcn― contanting o対 dizcd lipid dcrivativcs.ノ Bあ ノCあ′7771994;269:26066‑26075

P″

Supporting informatヨ on
AdditiOnal supporting infc9rlllation lnav bc fOund ill thc Onlinc vcrsioll of this articlc:

Figo Sl Schcnlatic diagraln Ofthc selectivc nitric Oxidc scnsOr(ll「 Ord dOCunlent)
Figo S2 1′ ′ノ∫′″visualizatiOn of nitric oxidc by 4,5‑dianlin01uOrcsccin(liacctatc staining cnrord docuincnt)

Figo S3 A Seruin leve1 0f asPartatC anlinotral〕 sfcrasc ancl alaninc■ lninOtrallstcrtlsc 6 h aftcr the tifth pcriOd Of
ischacnlin B Hist010野 :samples hal■ cstcdそ licr thc l■ hl)criOd ofischacll)in(ヽ VOrd dOcuincnt)

Figo S4 A Collccpttlal dingranl of the llitric oxide(NO)CurvC■ 1lal〕 7sis.B Arca ulldcr thc curc Of N0 1cvel du面 ng
cach ischacinic Pcriod.C Pcak diflcrcntial cocf6ciclll■ t CaCh tinlc point(1ヾ Ord dOCunlcnt)

PIcase nOtc:John lⅥ lev ct― Solls Ltd is nOt resPollsiblC br thc ll11lctiOnaliw Of ally supporting matcrials supplicdby thc authOrs.Ally qucl・ ics(otllCr than inissillg lllatcrial)should bC clircctcd tO the corrcspondingそ luthor fOr tllc
articlc.

t'2012 BriishJournal ofSurge,Socie、
Pul〕 lished byJolln、 viley&SOns

,

Ltd

Ltd

、、、=tls co uk