Intranasal post-cardiac arrest treatment with orexin-A facilitates arousal from coma and ameliorates

20 Pages·2017·5.04 MB·English

RESEARCHARTICLE
Intranasal post-cardiac arrest treatment with
orexin-A facilitates arousal from coma and
ameliorates neuroinflammation
HirenR.Modi1,QihongWang1,SahithiGD1,DavidSherman1,ElliotGreenwald1,Alena
V.Savonenko2,RomergrykoG.Geocadin3,NitishV.Thakor1*
1 DepartmentofBiomedicalEngineering,TheJohnsHopkinsUniversitySchoolofMedicine,Baltimore,
Maryland,UnitedStatesofAmerica,2 DepartmentsofPathologyandNeurology,TheJohnsHopkins
UniversitySchoolofMedicine,Baltimore,Maryland,UnitedStatesofAmerica,3 Neurologyand
a1111111111
Anesthesiology/CriticalCareMedicine,TheJohnsHopkinsUniversitySchoolofMedicine,Baltimore,
a1111111111
Maryland,UnitedStatesofAmerica
a1111111111
a1111111111 *[email protected]
a1111111111
Abstract
Cardiacarrest(CA)entailssignificantrisksofcomaresultinginpoorneurologicaland
OPENACCESS
behavioraloutcomesafterresuscitation.Significantsubsequentmorbidityandmortalityin
Citation:ModiHR,WangQ,GDS,ShermanD, post-CApatientsarelargelyduetothecerebralandcardiacdysfunctionthataccompanies
GreenwaldE,SavonenkoAV,etal.(2017)
prolongedwhole-bodyischemiapost-CAsyndrome(PCAS).PCASresultsinstronginflam-
Intranasalpost-cardiacarresttreatmentwith
orexin-Afacilitatesarousalfromcomaand matoryresponsesincludingneuroinflammationresponseleadingtopooroutcome.Cur-
amelioratesneuroinflammation.PLoSONE12(9): rently,therearenoprovenneuroprotectivetherapiestoimprovepost-CAoutcomesapart
e0182707.https://doi.org/10.1371/journal.
fromtherapeutichypothermia.Furthermore,therearenoacceptableapproachestopro-
pone.0182707
motecorticalorcognitivearousalfollowingsuccessfulreturnofspontaneouscirculation
Editor:JohannesBoltze,FraunhoferResearch
(ROSC).Hypothalamicorexinergicpathwayisresponsibleforarousalanditisnegatively
InstitutionofMarineBiotechnology,GERMANY
affectedbyneuroinflammation.However,whetheractivationoftheorexinergicpathwaycan
Received:March20,2017
curtailneuroinflammationisunknown.Wehypothesizethattargetingtheorexinergicpath-
Accepted:July24,2017 wayviaintranasalorexin-A(ORXA)treatmentwillenhancearousalfromcomaand
Published:September28,2017 decreasetheproductionofproinflammatorycytokinesresultinginimprovedfunctionalout-
comeafterresuscitation.WeusedahighlyvalidatedCAratmodeltodeterminetheeffects
Copyright:©2017Modietal.Thisisanopen
accessarticledistributedunderthetermsofthe ofintranasalORXAtreatment30-minutepostresuscitation.At4hrspost-CA,themRNAlev-
CreativeCommonsAttributionLicense,which elsofproinflammatorymarkers(IL1β,iNOS,TNF-α,GFAP,CD11b)andorexinreceptors
permitsunrestricteduse,distribution,and
(ORX1RandORX2R)wereexaminedindifferentbrainregions.CAdramaticallyincreased
reproductioninanymedium,providedtheoriginal
proinflammatorymarkersinallbrainregionsparticularlyintheprefrontalcortex,hippocam-
authorandsourcearecredited.
pusandhypothalamus.Post-CAintranasalORXAtreatmentsignificantlyamelioratedthe
DataAvailabilityStatement:Alldataarepublished
CA-inducedneuroinflammatorymarkersinthehypothalamus.ORXAadministration
hereandtherelevantdataarewithinthepaperand
itsSupportingInformationfiles.Theadditionaldata increasedproductionoforexinreceptors(ORX1RandORX2R)particularlyinhypothala-
regardingpublicationmaybeaskedto mus.Inaddition,ORXAalsoresultedinearlyarousalasmeasuredbyquantitativeelectro-
correspondingauthor.
encephalogram(EEG)markers,andrecoveryoftheassociatedbehavioralneurologic
Funding:ThisresearchwassponsoredbyNHLBI, deficitscalescore(NDS).OurresultsindicatethatintranasaldeliveryofORXApost-CAhas
NIHgrantR01HL071568,https://projectreporter.
ananti-inflammatoryeffectandacceleratescorticalEEGandbehavioralrecovery.
nih.gov/project_info_description.cfm?aid=
9085348&icde=33528322&ddparam=&ddvalue=
&ddsub=&cr=1&csb=default&cs=ASC&pball=.
PLOSONE|https://doi.org/10.1371/journal.pone.0182707 September28,2017 1/20
Orexin-Atreatmentreducespost-CAneuroinflammation
Competinginterests:Theauthorshavedeclared BeneficialoutcomesfromintranasalORXAtreatmentlaythegroundworkfortherapeutic
thatnocompetinginterestsexist. clinicalapproachtotreatingpost-CAcoma.
Abbreviations:ABG,arterialbloodgases;IQ,
InformationQuantity;NDS,neurologicdeficit
score;ORXA,Orexin-A;ROSC,returnof
spontaneouscirculation;TH,Therapeutic
hypothermia.
Introduction
Cardiacarrest(CA)entailssignificantriskofcomaordisordersofconsciousnessresultingin
poorneurologicaloutcomeafterresuscitation[1,2].Therapeutichypothermiahasbeenshown
toprovidesomebenefitsinpost-CAsurvivalandimprovedneurologicaloutcomesinclinics
andpreclinicalstudies[3–5][6].However,recentfindingsfromTargetedTemperatureMan-
agement(TTM)trialcontesttheviewthathypothermia(33˚C)wouldofferanadvantageover
36˚C[6].Whiledeephypothermiamaystillholdpromiseofbeneficialeffects,thereisan
unmetneedtoexplorealternativeapproachestofacilitatearousalandneuroprotection[7,8].
Theorexinergicpathwayhasemergedasanimportantpartofthebrain’sarousalsystem[7,9].
Thebrainstemanddiencephalicnucleimodulatedbytheorexinergicsystemregulatenotonly
arousalbutalsobasicphysiologicfunctions[10,11].Itoriginatesinthehypothalamusand
deliversorexintodifferentbrainregions.Orexin-A(ORXA)isaneuropeptidethatisresponsi-
bleformaintainingwakefulnessandiscloselyassociatedwithotherneurotransmitterpath-
ways[9,12,13].
GlobalcerebralischemiainducedbyCAandfollowedbyreperfusiontriggersamultitude
ofprocessesthatultimatelyresultinneuroinflammatoryresponseswithactivationofglial
cells,releaseofproinflammatorycytokines,anddelayedneuronaldeath[14–17].LevelsofIL-6
andTNF-αwereshowntobestronglyassociatedwithseverityofpost-CAsyndrome(PCAS)
[18],mortalityrate,andneurologicoutcomes[19–22].Importantly,thesystemicinflamma-
toryresponseafterCAwasnotmodifiedbytherapeutichypothermiaat33˚Cor36˚C.Despite
thepathophysiologicsignificanceofCA-inducedinflammationingeneralandneuroinflam-
mationinparticular[18,20–23],thepotentialofanti-inflammationtherapeuticapproachesin
limitingconsequencesofCAhasnotbeenadequatelystudied.Recentstudieshavesuggested
thatORXA,inadditiontoitswell-establishedroleinpromotingarousal[7,9],mayalsoexhibit
anti-inflammationproperties[24–27].CombinationofsucheffectsmakesORXAaparticular
attractivecandidateforthetreatmentofCA-associatedcomaandneuroinflammation.
OurpreviousworkexploredORXAasadrugthatmaypromotearousalinanexperimental
settingofCA-inducedcoma[12].Asingleintracerebroventricular(icv)injectionofORXA
resultedinanimprovementintheEEG-basedmeasureofarousalat4hrspostCA.However,
inthepreviousworkwedidnottestwhetherpost-CAORXAtreatmentcouldresultinany
detectablechangesincytokineproductioninthebrainthatwouldsupporttheideaofitsanti-
inflammatoryproperties.Inthepresentstudy,wehavechangedtherouteofadministrationto
increaseapotentialtranslationalvalueofthepost-CAORXAtreatment.Weusedanintranasal
deliveryofORXAthatcanbeeasilyimplementedinclinicalsettings.Inthisproof-of-concept
studywehaveoptedtotestbeneficialeffectsofORXAatthetimepointatwhichtheicvORXA
treatmenthasalreadybeenproveneffective(4hrs)[12].WehaveaccessedORXAeffectson
neurologicalarousalbymeansofquantitativeEEGthatinourpreviousstudieshasbeensensi-
tivetoseverityofCA-associatedinjuryaswellaspredictiveofbehavioraloutcomesafterhypo-
thermia[28,29]andicv-ORXAtreatment[12,30].ThedevelopmentofourquantitativeEEG
approachesduringthepreviousstudieshassuggestedthatanEEG-derivedgammafrequency
bandmightbeaparticularpromisingtoolforassessingneurologicalarousal.Behavioralout-
comeswereanalyzedbyaneurologicaldeficitscore(NDS)[31,32]thatisawell-accepted
PLOSONE|https://doi.org/10.1371/journal.pone.0182707 September28,2017 2/20
Orexin-Atreatmentreducespost-CAneuroinflammation
standardtocharacterizeeffectsofCA[33–35]andhasbeenusedinourpreviousstudyof
post-CAORXAtreatment[12].
Inthecurrentstudy,wehavetestedthehypothesisthatintranasaldeliveryofORXAwill
improveneurologicalandbehavioraloutcomesofCA-inducedcoma.Anadditionalimportant
focusofourstudyistheexplorationofthepathwaysinvolvedinORXAaction.Wehypothe-
sizethatORXAwillmitigatetheneuroinflammatoryresponse.Thegoalsofourstudyareto
demonstratetheeffectsofintranasalORXAtreatmentonfacilitatingarousalfromcoma(as
judgedbybehavioralandcorticalEEGoutcomes),and,inaddition,ameliorateneuroinflam-
matoryresponsesinabrainregion-specificmanner.Thesestudiesshouldserveasaproof-of-
conceptforintranasaldeliveryofORXA,andshouldgiveanimpetustofurtherclinicaldevel-
opmentofthetreatmentsthatsuccessfullycombinepro-arousalandanti-inflammatoryeffects.
Materialsandmethods
Animalmodel
Subjectsandimplantationofelectrodes. AdultmaleWistarrats,(300-350g;Charles
River,Wilmington,MA)wereutilizedforthisproject.Thestudywasdesignedasaproof-of-
conceptexperimentforintranasaladministrationofORXA.Ourpreviousexperimentswithicv
deliveryofORXAwereconductedinmales[12],sowehavechosentouseanimalsofthesame
sextoavoidpossibleinterferenceofanadditionalfactor(sex)withtheeffectsofintranasal
ORXA.AllexperimentalprocedureswereapprovedbytheInstitutionalAnimalCareandUse
CommitteeattheJohnsHopkinsUniversitySchoolofMedicine.Theanimalswerehabituated
for1weekbeforeelectrodeimplantation.Electrodesimplantationwasconductedunder2–3%
isofluraneanesthesiadeliveredviaamask.Ratswereimplantedwith5epiduralEEGscrewelec-
trodes(PlasticsOne,USA),withtwooftheelectrodesplacedat2mmanteriortoBregmaand
2mmtotherightandleftofBregma,correspondingtotheM1regionsofthefrontalcortex.
Additionaltwoelectrodeswereplacedat6mmposteriortoBregmaand4mmtotherightand
leftofBregma,correspondingtotheV1regionsoftheoccipitalcortex.Aground/referenceelec-
trodewasplacedoverthecerebellarregion(2mmposteriortoLamda).Theelectrodeswere
attachedtoapedestal(MS363,PlasticsOne,USA)andstabilizedwithdentureresin(LangDen-
tal,USA)tofacilitateconnectionwiththeneuralrecordingdevice,anRX5TDTdevice(Tucker
DavisTechnologies,Alachua,FL).Theelectrodeswereimplanted1weekpriortoasphyxia-
inducedCAexperimentstoallowfullrecoveryfromthispreliminarypreparation[12,30].
Asphyxial-CAmodel. Oneweekafterelectrodeimplantation,animalsweresubjectedto
asphyxia-induced7minCA(Fig1).Basedonthepriorstudies[12,30,36],wehaveexpected
thatthisdurationresultsinaninjurythatisbroadandsignificantbutstillresultsinthesur-
vivaloftheanimalpostCA,andthusallowingexaminationandtitrationofneuroprotective
strategies.OurpreviousstudyhasalsodemonstratedthatCAinthismodelresultsinneuronal
injurywhentestedasearlyas4hrspost-ROSC[37].PriortoCA,ratswereendotracheallyintu-
batedbydirectlaryngoscopyandmechanicallyventilatedwith2%isofluranein50%O +50%
2
N gas,afterwhichthefemoralarteryandveinwerecannulatedwithpolyethylene50tubing
2
catheterstomonitorbloodpressure(BP)andsamplearterialbloodgases(ABGs)andto
administerintravenousmedications.OurCAprotocolhasbeendescribedindetailpreviously
[12,30,36].Briefly,wecarriedout10minutesofbaselineEEGrecordingunderisoflurane.Fol-
lowingbaselinerecording,anesthesiawaswashedoutforatotal5minutes,startingwith2
minutesof100%O withoutisofluranetocapturenon-anesthetizedEEG.Thenfor3minutes,
2
FiO wasdecreasedto20%with80%ofN gas(roomair).Rocuroniumbromide2mg/kgIV
2 2
wasadministeredformuscleparalysisat2minutesofwashouttime.Following5minutesof
gaswashout,globalasphyxiawasinducedbystoppinganddisconnectingtheventilatorand
PLOSONE|https://doi.org/10.1371/journal.pone.0182707 September28,2017 3/20
Orexin-Atreatmentreducespost-CAneuroinflammation
Fig1.Schematicdiagramofexperimentaldesign.
https://doi.org/10.1371/journal.pone.0182707.g001
clampingthetrachealtube.Globalasphyxiawasaccompaniedbytransienthypertension,fol-
lowedbyprogressivebradycardia,hypotensionandeventualCA(definedbyMAP<10mmHg
andcessationofelectricalrhythmandnon-pulsatile-pressurewave)areobserved.
Resuscitation
AfterthepredeterminedCAperiod(7min),cardiopulmonaryresuscitation(CPR)wasiniti-
atedbyunclampingthetrachealtube,restartingmechanicalventilationwith100%O ,
2
PLOSONE|https://doi.org/10.1371/journal.pone.0182707 September28,2017 4/20
Orexin-Atreatmentreducespost-CAneuroinflammation
administeringepinephrine(5μg/Kg,i.v.),andapplyingsternalchestcompressionswithtwo
fingers(~200compressions/min,togeneratemeanarterialpressure(MAP)>50mmHg)and
NaHCO3(1mmol/Kg,i.v.)tonormalizearterialpH.Whentheaforementionedmeasureswere
achieved,theresultingtimewasnotedasareturnofspontaneouscirculation(ROSC).ABGs
weresampled15minutesafterROSC.Anesthesiawasnotprovidedpost-resuscitationinorder
tominimizedrugeffectsonrecordedneural(EEG)signalsandtofacilitatethereturnof
arousal.Followingsuccessfulresuscitation,theanimalwashyperventilated(TV10ml/Kg,RR
65/minandPEEP3cmH O)for10min.Subsequentventilatorchangeswereperformed
2
accordingtotheABG.RRwasadjustedto50/min[12,36].Rectaltemperaturewasmaintained
at37.0˚Cusingawarmingpadandlamp.Oncearatachievedspontaneousrespirations,itwas
extubated,andvascularcatheterswereremoved,sutureswereadministeredtothefemoral
region.
TreatmentwithOrexin-A(ORXA)
At30minutespost-ROSC,theratswererandomizedtoreceivesaline(vehicle),low(10μM)
ORXAorhighdose(50μM)ofORXAintranasally.Eachanimalreceived10μlx3ineachnos-
trilfora30secondinterval(60μltotal).ThelowdoseORXAgroupreceived600nmoles
(10μM/L=10nmoles/1μL;10nmolesX60μl=600nmoles)andhighdose(50μM)ORXA
groupreceived3000nmolestotal,respectively.
Behavioraltesting
TheNeurologicDeficitScalescore(NDSscore),whichwaspreviouslydevelopedandvalidated
extensively[31,32,37]rangesfrom0–80andservesasasurrogatequantitativerodentneurode-
ficitandcomascore,analogoustohumancomascores.TheNDSwaspreviouslyusedtoquan-
titaterodentcomaandarousallevels[36,38].TheNDSassessesarousal,cranialnervereflexes,
andmotorbehavior.SeeS1Tablefordetailsofitscomponents.TheNDSwasdeterminedat4
hourspost-ROSC.Trainedpersonnelwhowereblindedtothevehicleandtreatmentgroups
conductedNDSexaminations(S1Table).
EEGrecordingandanalysis
BeforeperformingCA,baselineEEG(15minutes)wasrecorded.Thesignalsweredigitalized
usingthedataacquisitionpackageCODAS(DATAQInstrumentsINC.,AkronOH).EEGwas
monitoredfor4hourspost-CAcontinuously.TheEEGwasdownsampledfrom12,200sam-
plespersecondto122.EEGgammaband(γ=30-50Hz)energywascalculatedusingtheTea-
gerEnergyOperator[39].Additionally,delta(1–3Hz),theta(4–7Hz),alpha(8–12Hz)and
beta(21–30Hz)energybandswerealsocalculatedforeverysample.TheGammaFractionwas
obtainedbydividingthegammabandenergybythetotalpowerunder50Hz.Thesemeasure-
mentswerethenaveragedoveroneminute.GammabandEEGwaspreviouslyvalidatedasa
measureofarousalfromcoma[30,36,38,40].
Brainsamplescollections
FourhoursafterCAandresuscitation,theanimalwasanesthetizedwithisofluraneandthen
decapitated.Thebrainwasrapidlyexcisedandthefrontalcortex(PFC),caudalcortex,hippo-
campus,striatum,hypothalamus,medulla,restofthebrain(ROB),andcerebellumweredis-
sected.Eachbrainregionwasfrozenin2-methylbutaneat−50˚C,andstoredat−80˚Cuntil
use.
PLOSONE|https://doi.org/10.1371/journal.pone.0182707 September28,2017 5/20
Orexin-Atreatmentreducespost-CAneuroinflammation
TotalRNAisolationandrealtimeRT-PCR
Ourchoiceofproinflammatorymarkerswasmainlybasedontwocriteria:whetherthemark-
ersareactivatedintheearlyphaseofinflammationandhowwelltheyarecharacterizedinthe
modelsofischemia.Bythesecriteria,IL-1β,TNFα,andiNOSareallinvolvedinanacute
phaseofinflammationandarewidelyusedmarkersofneuroinflammationinducedbyische-
mia[14,41].GFAPwasusedasamarkerofastrocytesandincreasesinGFAPtranscription
couldbeconsideredasamarkerofinitiationofastrogliosis.CD11bhasbeenchosenasa
widelyusedmarkerofmicroglialactivationinthebraininsettingsofproinflammatory
responses.TotalRNAwasisolatedfromdifferentbrainstructuresusinganRNeasylipidtissue
minikit(Qiagen,Valencia,CA).Briefly,thetissuewashomogenizedinQiagenlysissolution
andtotalRNAwasisolatedbyphenol-chloroformextraction.ComplementaryDNAwaspre-
paredfromtotalRNAusingahigh-capacitycDNAArchivekit(AppliedBiosystems,Foster
City,CA).mRNAlevels(IL1β,iNOS,CD11b,TNF-α,GFAP,ORX1R,ORX2R)weremea-
suredbyquantitativeRT-PCR,usinganABIPRISM7000sequencedetectionsystem(Applied
Biosystems).Specificprimersandprobesforthesemarkers(S2Table),purchasedfromTaq-
ManRgeneexpressionassays(AppliedBiosystems),consistedofa20XmixofunlabeledPCR
primersandTaqmanminorgroovebinder(MGB)probe(FAMdye-labeled).Thefold-change
ingeneexpressionwasdeterminedbytheΔΔCTmethod[42].Dataareexpressedastherela-
tivelevelsofthetargetgeneintheCAratnormalizedtotheendogenouscontrol(GAPDH)
andrelativetothecontrol(shamcontrol).Allexperimentswerecarriedoutinduplicatewith
10shamcontrols,6brainsamplesforCA+salineand6brainsamplesfromCA+ORXAtreat-
mentrats.Dataareexpressedasrelativefoldchangeingeneexpression.
Statistics
Dataarepresentedasmean±SEM.Whenthreegroupswerecompared(shamcontrol,saline
treatedCAandORXtreatedCA),statisticalsignificancewasdeterminedusingmaineffector
mixeddesignANOVAsfollowedbypost-hoctestswhenappropriateforcomparisonsbetween
particularsetofmeans.Statisticalsignificancewassetatp(cid:20)0.05.Plevelsformultiplecompar-
isonsweremodifiedbyBonferronicorrection.Detailsofstatisticalanalysesforbehavioraland
EEGdataarepresentedinS3,S4andS5Tables,respectively.Resultsofstatisticalanalysesfor
mRNAlevelsofcytokinesandORXreceptorlevelsindifferentbrainstructuresarepresented
inS6–S9Tables.
Results
BaselinecharacteristicsofORXAtreatedandcontrolsalineanimals
Weobtainedbaselinecharacteristicsofbodyweight,bloodgasesandhemoglobinlevelsforall
ratsimmediatelypriortoCA(Table1).Nostatisticaldifferenceswereobservedforthese
parametersbetweengroups,confirmingsuccessfulrandomizationintothetreatmentgroups
(Table1).Baselinehemodynamicparameters,includingheartrate(HR)andBPmeasurements
asobtainedbyarteriallinemeasurementspriortoORXAorsalinetreatmentdidnotshowany
significantdifferencesaswell(Table2).
CAinducedcoma
AsillustratedinTable1,timestoenterCAorreturnofspontaneouscirculation(ROSC)were
statisticallyequivalentbetweengroups.InourCAmodel(7min-longCA),thefailureto
achieveROSC(failureofresuscitation)isabout10%.AfterthisprotocolofCA,allanimals
remainedincoma.Theyusuallystartedregainingalertnessaround4hrspost-CA.
PLOSONE|https://doi.org/10.1371/journal.pone.0182707 September28,2017 6/20
Orexin-Atreatmentreducespost-CAneuroinflammation
Table1. Baseline(pre-cardiacarrest)characteristicsofOrexin-Aandcontrolgroups. Nosignificant
differencesinbaselinecharacteristicsbetweenanimalsinCA+Saline(n=6)andCA+Orexin-A(50μM)
(n=6).
Parameters CA+Saline CA+Orexin-A
(n=6) (n=6)
Weight(g) 367.4±7.59 365.12±3.11
pH 7.44±0.12 7.37±0.14
pCO2(mmHg) 40.05±1.74 39.12±2.39
pO2(mmHg) 162.2±8.18 177.66±16.43
HCO3(mmol/L) 27.52±0.57 29.33±0.46
Na(mmol/L) 136.93±0.43 136.00±0.40
Hemoglobin(mmol/L) 12.01±0.25 12.33±0.09
TimetoCA(s) 243.33±4.81 241.5±8.54
TimetoROSC(sec)* 96.33±13.14 102.5±6.76
Groupmeans±SEMareshown.
*ROSCindicatesreturnofspontaneouscirculation.
https://doi.org/10.1371/journal.pone.0182707.t001
HemodynamicresponsetoORXAtreatment
Hemodynamicmeasurementsinthesaline-treatedratsrevealedsignificantdecreasesinheart
rateandbloodpressurebetween30and60minafterROSC(Fig1;Table2).Incontrasttothe
salinegroup,theORXA-treatedratsshowedmorestableparametersofhemodynamicsduring
thistimeperiod(Table2).ItisnoteworthythatthiseffectofORXAtreatmentwaslimitedto
onlybloodpressurebutnotheartrate(Table2).
Orexin-Aimprovesbehavioralarousal(NDS)
Neurologictestingofratswasconductedat4hrspost-ROSCinbothsalineandORXAtreated
groups(Fig2).Toanalyzeadose-responseofbehavioraleffectsofORXA,twoexperimental
Table2. Hemodynamicparametersat30minutespost-ROSC(beforedrug)and30minutepostOrexin-Atreatment. Nosignificantdifferenceswere
observedbeforetreatments.TheeffectsofORXA(50μM)orSalineweremeasured30minafterthetreatment.
GROUP 30minutesPost-ROSC 30minutesPost-Drug
(Pre-TreatmentDrug) (Post-TreatmentDrug)
CA+Saline CA+Orexin-A CA+Saline CA+Orexin-A
(n=6) (n=6) (n=6) (n=6)
SBP 130.93±6.14 143.66±10.48 106.32±7.95# 130.51±6.74
(mmHg)
DBP 86.90±4.04 88.32±6.79 74.25±6.38# 86.73±5.80
(mmHg)
MAP 101.64±4.67 106.77±7.93 86.49±6.77*# 106.63±6.37
(mmHg)
Pulse 380.23±6.60 373.82±23.07 340.06±11.52# 336.42±10.86#
(bpm)
Groupmeans±SEMareshown.ROSCindicatesreturnofspontaneouscirculation;SBP,systolicbloodpressure;DBP,diastolicbloodpressure;andMAP,
meanarterialpressure;bpm,beatsperminute.
Two-waymixeddesignANOVAs(GroupxTimepoint)wereusedforstatisticalanalyses.Anasterisk(*)indicatesasignificantbetween-groupdifferenceat
thepost-drugperiod,p<0.05;pound(#)indicatesignificanteffectsofTimepoint(Post-ROSCvsPost-Drug)inthesamegroupofanimals,p<0.05
(Neuman-Keulspost-hoctestappliedtoasignificantANOVAmaineffectorinteraction).
https://doi.org/10.1371/journal.pone.0182707.t002
PLOSONE|https://doi.org/10.1371/journal.pone.0182707 September28,2017 7/20
Orexin-Atreatmentreducespost-CAneuroinflammation
Fig2.Intranasalorexin-AtreatmentimprovesbehavioraloutcomesafterCA.(A)NeuroDeficitscore(NDS)wastestedat4hrsafterROSC
forcontrol(CA+saline)(n=6),CA+10μMorexin-A(n=6),andCA+50μMorexin-A(n=6)treatmentgroups.(B)Schematicrepresentationof
correlationsbetweenindividualNDSsubscores.SeeS3Tableforcorrelationcoefficients.(C)SubscoresofCluster1wereimprovedaftertreatment
with50μMORXA.Valuesarereportedasmean±SEM.SingleanddoubleasterisksindicatesignificantdifferencesfromSalinegroupasaresultof
LSDpost-hoctestwithplevelslessthan0.025or0.0001,respectively.SingleanddoublepoundsignsindicatesignificantdifferencesfromORXA-
10groupasaresultofBonferronipost-hoctestwithplevelslessthan0.025or0.0001,respectively.SeeS2Tablefortheresultsofappropriate
ANOVAs.
https://doi.org/10.1371/journal.pone.0182707.g002
groupswereincluded(ORXA-10μMandORXA-50μM).TotalNDSscoreconsistsofsum
from18differenttestswiththemaximumof80points(bestperformance)andtheminimum0
points(worstperformance)(S1Table).At4hrsafterROSC,comparisonoftotalNDSscores
betweenthesaline,ORXA-10andORXA-50groupsrevealedbetterperformanceintherats
treatedwith50μMofORXAascomparedwithtwoothergroups(Fig2A,S2Table).Lower
doseofORXA(10μM)failedtoshowefficacyastherewerenosignificantdifferencesbetween
thesalineandORXA-10groups.SinceNDStestingisdesignedtoreflectafullrangeofbehav-
ioralrepertoirefromarousaltocoma,notallcategoriesofbehavioralresponsesaresensitiveat
particularstatesofarousal.Inthenextsetofanalyses,weremovedallbehavioralresponses
thatshowednovariabilitydueto“floor/ceiling”effects.Thisyieldedasetof12variables.To
revealpossiblecorrelationswithinthissetofvariablesthedataweresubmittedtoSpearman
RankOrdercorrelations(S3Table).Thisanalysisrevealedtwoclustersofcorrelatedvariables
(Fig2B).Cluster1includedNDSsubscoresofalertness,vision,painreactivity,strengthand
acousticstartlereaction,whileCluster2reflectedolfaction,swallowing,andreactiontowhis-
kerstimulation(Fig2B).Thepupillaryandrightingreflexesaswellasindexesofrespiration
andseizureswerenotcorrelatedtoanyothersubscores(Fig2B).Analysisoftheeffectof
ORXAtreatmentsonNDSsubscoresrevealedasignificantimprovementinbehavioralout-
comesduetoanincreaseinsubscoresofCluster1aftertreatmentwith50μMORXA(Fig2C,
S2Table).Therewerenosignificantimprovementsforthe10μMORXAtreatmentgroup.
Basedonbehavioroutcomeresults(NDSresults)post-CA,wetookthisstudyforward
using50μMORXAtreatmenttoevaluateneurophysiologicalarousalanditsanti-inflammatory
effectinthebrain.
Orexin-ApromotesarousalafterCA/resuscitationasmeasuredbyEEG
gammafraction
Next,weinvestigatedtheeffectofintranasal50μMORXAonEEGgammabandpower(30–50
Hz),ameasureofneurophysiologicarousalinthebrain.TheEEGgammabandhasalsobeen
PLOSONE|https://doi.org/10.1371/journal.pone.0182707 September28,2017 8/20
Orexin-Atreatmentreducespost-CAneuroinflammation
showntobeapredictorofgoodneurologicaloutcome[43].EEGgammapowerwasindistin-
guishablebetweengroupsbeforetreatments(Fig3A–3C,S4Table)supportingasuccessful
randomizationintothegroups.Aftertreatments,theORXA-50groupdemonstratedsignifi-
cantlyhigherEEGgammapowerwhentestedduring~3.5hrspost-treatmentperiod(Fig3A
and3D,S4Table).SignificanteffectofORXA-50wasrevealedmainlyduetodifferences
observedfrom45to165minposttreatment(Bonferronipost-hoctest,p<0.05)(Fig3A).At
Fig3.EEGIQgammaband(30–50Hz)forthe50μMOrexin-Agroup(redline,n=6)andthegroupofcontrolratstreatedwithsaline
(blackline,n=6).(A)Sub-bandEEGgammapowertracingshowsthatorexin-AleadstoimprovedEEGIQshortlyafterintranasal
administrationingammabandEEGIQlevels.(B-D)EEGgammapoweraveragedforperiodsofbaselinerecording(B),ROSC(C),andpost-
treatment(Saline/ORXA)(D).Dataarepresentedinmean±SEM.BLindicatesbaseline(pre-anesthesia);CA:asphyxialcardiacarrest;Drug:
representstheperiodaftersalineororexintreatment.Asteriskindicatessignificantbetween-groupdifferencesforthepost-drugperiod,
p<0.021.ResultsofstatisticalanalysesarepresentedinS4Table.
https://doi.org/10.1371/journal.pone.0182707.g003
PLOSONE|https://doi.org/10.1371/journal.pone.0182707 September28,2017 9/20
Orexin-Atreatmentreducespost-CAneuroinflammation
latertimepointsthedifferencesbetweenthesalineandtheORXA-50groupsdisappeareddue
torecoveryofEEGgammabandinsalinetreatedanimals.Thus,intranasaltreatmentwith
50μMORXAimprovedearlyrecoveryofEEGgammapower,particularlyinthefirst2.5hrs
afterthetreatment.
Post-CAOrexin-AtreatmentprimarilyincreasesmRNAlevelsofORX
type1receptor
Therearetwotypesofreceptorstoorexin,andORXAneuropeptidecanbindandactivate
bothorexinreceptor1(ORX1R)andorexinreceptor2(ORX2R)[44].mRNAlevelsofORX
receptorsarecharacterizedbydramaticvariabilitybetweendifferentbrainstructureswiththe
maximumlevelsdetectedinthehypothalamusforbothtypesofreceptors(Fig4A).CA-
inducedchangesinmRNAlevelsofORX1Rwerealsodependentonabrainstructureandvar-
iedfromactivation(PFC,Medulla)toinhibition(CaudalCortex,Hypothalamus,Cerebellum)
(Fig4B).Predominanteffectofpost-CAtreatmentwithORXAwasanincreaseinORX1R
mRNAlevelsascomparedtocontrolorCA-Salinegroup(Fig4B).IncontrasttoORX1R,
ORX2RmRNAlevelsweremostlydecreasedasaresultofCAand/orORXAtreatment.The
onlyexceptionwasthehypothalamuswhere,inconcertwithORX1R,mRNAlevelsofORX
Fig4.EffectsofCAandORXAtreatment(50μM)onmRNAlevelsofORXreceptorsindifferentbrain
structures.(A)mRNAlevelsofOrexinreceptor1and2indifferentbrainstructuresascomparedtoCerebellum.
(B-C)EffectsofCAandORXAtreatmentonmRNAlevelsofORXreceptor-1(B),andORXreceptor-2(C).
Resultsareexpressedrelativetothecontrolgroup.Asterisksandpoundsingsindicatesignificantdifferences
fromShamcontrolorCA+Salinegroup,respectively,asaresultofLSDpost-hoctestswithplevelscorrectedfor
multiplecomparisons,p <0.0021(seeS5TableforresultsofANOVA).ProbesequencesforRT-PCRare
cor
presentedinS2Table.
https://doi.org/10.1371/journal.pone.0182707.g004
PLOSONE|https://doi.org/10.1371/journal.pone.0182707 September28,2017 10/20