The Effects of Rilmenidine and Perindopril on Arousal Blood Pressure during 24 Hour Recordings

19 Pages·2016·2.08 MB·English

RESEARCHARTICLE
The Effects of Rilmenidine and Perindopril on
Arousal Blood Pressure during 24 Hour
Recordings in SHR
KyungjoonLim1*,KristyL.Jackson1,SandraL.Burke1,GeoffreyA.Head1,2
1 NeuropharmacologyLaboratory,BakerIDIHeartandDiabetesInstitute,Melbourne,Victoria,Australia,
2 DepartmentofPharmacology,MonashUniversity,Clayton,Victoria,Australia
*[email protected]
Abstract
Thesurgeinarterialpressureduringarousalinthewakingperiodisthoughttobelargely
a1111111111
a1111111111 duetoactivationofthesympatheticnervoussystem.InthisstudywecomparedinSHRthe
a1111111111 effectsofchronicadministrationofthecentrallyactingsympatholyticagentrilmenidinewith
a1111111111 anangiotensinconvertingenzymeinhibitorperindoprilontherateofriseandpowerofthe
a1111111111
surgeinmeanarterialpressure(MAP)thatoccurswitharousalassociatedwiththeonsetof
night.Recordingsweremadeusingradiotelemetryin17adultSHRbeforeandaftertreat-
mentwithrilmenidine(2mg/kg/day),perindopril(1mg/kg/day)orvehicleinthedrinkingwater
for2weeks.RilmenidinereducedMAPby7.2±1.7mmHgwhileperindoprilreducedMAP
OPENACCESS by19±3mmHg.Doublelogisticcurvefitanalysisshowedthattherateandpowerof
Citation:LimK,JacksonKL,BurkeSL,HeadGA increaseinsystolicpressureduringthetransitionfromlighttodarkwasreducedby50%and
(2016)TheEffectsofRilmenidineandPerindopril 65%,respectively,buthadnoeffectondiastolicpressure.Rilmenidinealsoreducedblood
onArousalBloodPressureduring24Hour
pressurevariabilityintheautonomicfrequencyintheactiveperiodasassessedbyspectral
RecordingsinSHR.PLoSONE11(12):e0168425.
doi:10.1371/journal.pone.0168425 analysiswhichisconsistentwithreductioninsympatheticnervoussystemactivity.Perindo-
prilhadnoeffectontherateorpowerofthearousalsurgeineithersystolicordiastolicpres-
Editor:Germa´nE.Gonza´lez,Universidadde
BuenosAires,ARGENTINA sure.Theseresultssuggestthatthearousalinducedsurgeinbloodpressurecanlargelybe
reducedbyanantihypertensiveagentthatinhibitsthesympatheticnervoussystemandthat
Received:June29,2016
angiotensinconvertingenzymeinhibition,whileeffectiveinreducingbloodpressure,does
Accepted:November30,2016
notaltertherateorpowerofthesurgeassociatedwitharousal.
Published:December21,2016
Copyright:©2016Limetal.Thisisanopenaccess
articledistributedunderthetermsoftheCreative
CommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalauthorand Introduction
sourcearecredited.
Hypertensionisanimportantriskfactorforpredictingcardiovasculardiseasebutitisthe
DataAvailabilityStatement:S1Filecontains morningperiodthatistheperiodofgreatestriskofstrokeandmyocardialinfarcts[1–6].
entireminimaldataset. Duringthemorningperiodthereisagradualincreaseinbloodpressure(BP)associated
Funding:Theauthorsreceivednospecificfunding withthenormalcircadianpatterninhumansasBPmovestowardsitshigherdaytimelevel.
forthiswork. Thisprocessisnotasuddenjumpbuttakesseveralhours.Whilemethodsforanalysisof
diurnalchangesincardiovascularvariableshavenoteasilydeterminedtherateofchangein
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. BPduringdifferentperiodsoftheday,wehavedevisedanewmathematicalanalysiswhich
PLOSONE|DOI:10.1371/journal.pone.0168425 December21,2016 1/19
SympatheticNervousSystemContributiontoArousalBloodPressureSurge
canestimatetherateofchangeinBPandheartrate(HR)duringthetransitionsbetween
sleepandawake.Wehaveshownthathypertensivehumans[7]andrats[8]haveagreater
rateofriseinBPduringtheperiodofarousalfromsleepcomparedtonormotensives.We
havealsoshownthatthisgreaterrateofriseinBPisasignificantandindependentriskfactor
inhumans[9]andisrelatedtotheactivationofthesympatheticnervoussystem[10].Earlier
studiescomparedthefrequencyofcardiacsynchronisedsympatheticburstsintheperineal
nerveanddidnotshowadifferencebetweenthemorningandeveningperiod,suggesting
thattherewasnodifferencebetweensympatheticactivityintheseperiods[11].However,it
istheamplitudeoftheburstthatwefoundisrelatedtothemorningsurgeinbloodpressure
andnotthefrequencyoffiring[10].Importantlytheamplitudeofthesympatheticburstis
alsoelevatedinconditionssuchasexperimentalhypertensioninducedbyangiotensininfu-
sionandhypoxia[12].Theamplituderepresentstheactivityofonlyactivefibreswhich
undernormalconditionsisaminoritywiththemajoritybeing“silent”or“inactive”.An
increaseinburstamplitudethereforesuggeststhatpreviouslysilentfibresarebeing
recruitedtobecomeactive.Werecentlyconfirmedthatindividualsympatheticunitsdidnot
increasefiringrateinhypertension[13].Takentogether,thesefindingssuggestthatthe
morningsurgeinbloodpressurethatoccursduringarousalischaracterisedbyactivationof
newsympatheticfibres.
WhiletherateofriseinBPisclearlyimportant,themagnitudeoftherisealsohasconsider-
ableinfluenceontheimpactoftheriseinpressure.Indeedmostmeasuressuchasthatdevel-
opedbyKarioandcolleagueshaveusedanestimateofthemorningchangeinBPwithina
specifiedperiodofwaking[14].TermedthemorningBPsurge(MBPS),thismeasurehasbeen
extensivelyusedintheliterature.Wehaverecentlydevelopedanovelmeasureofthemorning
surgeinBPwhichwehavetermedthe“BP ”whichistheproductoftherateandthe
Power
amplitudeoftheBPmorningsurge[1].BPpoweris2.5foldgreaterinhypertensivesubjects
thanmatchednormotensivepatients[1]andmaythereforerepresentmoreeffectivelythe
impactofthemorningsurge[1].Werecentlycomparedtherateofrise,BPpowerandMBPS
withactivationofthesympatheticnervoussystemin35patientsandfoundthatthesympa-
theticburstamplitudewasmostrelatedtotheBP andrateofrisebutnotatalltheMBPS
Power
[10].ThuswehypothesisethatthemorningBP wouldbemostsusceptibletoattenuation
Power
withpharmacologicalagentsthattargetthesympatheticnervoussystemsuchascentrallyact-
ingantihypertensiveagents.Wehaveextensiveexperiencewithrilmenidineandmoxonidine
whicharesecondgenerationagentsofthisclassthathavemixedactionsonα -adrenoceptrors
2
andimidazolinereceptors[15].Theprincipleantihypertensiveeffectsofrilmenidineandmox-
onidinearethroughinhibitionofsympatheticactivity[16]andthisinvolvesmainlyactivation
ofimidazolinereceptorsintherostralventrolateralmedulla[17–19].Rilmenidineisalso
knowntofacilitatethecardiacbaroreflexthroughgreatervagalactivitybutonlyduringthe
lightinactiveperiodinmice[20].Wehavealsoobservedthatrilmenidinereducedtherateof
riseinBPthatoccursattheonsetofdarknessinhypertensivemice[20].However,wedonot
knowtheeffectofrilmenidineonthearousalBP norwhetherthisissimplyduetoa
Power
reductioninBPitself.
Hence,theaimofthepresentstudywastoapplythenewlogisticcurvefittingprocedureto
BPrecordingsfromratsinordertodeterminetheeffectsofchronicadministrationofthecen-
tralsympatholyticagentrilmenidineontelemetryrecordingsofBP,HRandlocomotoractivity
inspontaneouslyhypertensiverats(SHR).Inparticularwetestedthehypothesisthatthesym-
patholyticagentrilmenidinewouldattenuatetherateofriseinBPandtheBP during
Power
nightonsetarousal.Wealsocomparedthefindingswithanangiotensinconvertingenzyme
inhibitorperindopriltodeterminetheeffectofreducingBPbutnotaffectingtheSNS.
PLOSONE|DOI:10.1371/journal.pone.0168425 December21,2016 2/19
SympatheticNervousSystemContributiontoArousalBloodPressureSurge
MaterialsandMethods
Animals,surgicalprocedures
AllprocedureswereapprovedbytheAlfredHospital/BakerHeart&DiabetesInstituteAnimal
EthicsCommitteeandwerecarriedoutaccordingtoguidelinessetbytheNationalHealthand
MedicalResearchCouncilofAustralia.Eighteenmale12–14weekoldSHRwereobtained
fromtheBakerHeart&DiabetesInstitute,weighing341±11gattheonsetoftheexperiments.
Telemetryprobeimplantation
Inaninitialoperation,theratswereinstrumentedwitharadio-telemetryprobe(DataSci-
ences,MA,USA)aspreviouslydescribed[21].Theratswereanesthetizedwithhalothane
deliveredbyamaskfromanopencircuitvapourizer(Goldman)andtheabdominalaorta
exposedthroughamidlineincision.Thebloodpressuresensor(modelTA11PA-C40)was
insertedintotheaortaclosetothefemoralbifurcationandfixedwithadropoftissueglue.
Thebodyofthetransmitterwassuturedtotheinsideoftheabdominalmusclewall.Allinci-
sionswerethensuturedandtheratwasgivencarprofenforanalgesia(5mg/kg,Pfizer,NSW,
Australia)andallowedtorecoverfromanesthesiainawarmbox.Aftersurgery,theratswere
housedsinglywithfreeaccesstotapwaterandstandardratpelletchow.
Radiotelemetryrecordingmethod
Theradiotransmitter,onceimplantedandturnedonbyamagneticswitch,providedacontin-
uousmeasureofthearterialpressurewaveformaswellasameasureoflocomotoractivity
[21].TheAMradiosignalswereanencodedpulsepositionmodulatedserialbitstream,col-
lectedbythereceiver(modelRPC-1,DataSciencesInternational)placedunderneaththeani-
mal’scage.Thesignalsfromthereceiverwerepassedtoananalogueconverter(modelRP11A,
DataSciencesInternational).Ambientbarometricpressurewasalsomeasured(APR-1,Data
SciencesInternational)andsubtractedfromthetelemeteredpressurebythedatacollection
systeminordertocompensateforchangesinatmosphericpressure.Theanaloguevoltagesig-
nalwasthenconvertedbyananaloguetodigitalacquisitioncard(PCplus,NationalInstru-
ments,AustinTexas,USA)usingsoftware(Universalacquisition)writteninLabview
(NationalInstruments).
Experimentalprotocol
After1–2weeksofpost-operativerecovery,theratswerehousedinindividualboxeswitha
12-hourlight-darkrhythm(lightson7.00am,lightsoff7.00pm).FromtheBPsignal,analgo-
rithmwasusedtodetectsystolicarterialpressure(SAP),diastolicarterialpressure(DAP)and
pulseinterval[22].Meanarterialpressure(MAP)wascalculatedonabeattobeatbasisand
instantaneousHRwascalculatedfromthepulseinterval[22].Anindexoflocomotoractivity
wasobtainedbymonitoringchangesinthereceivedsignalstrength.Foreachheartbeat
detected,systolictime,SAP,MAPandDAP,pulseintervalandlocomotoractivitywerestored
intextformatonanIBM-compatiblecomputer.
Followinganinitial3-daycontrolmonitoringperiod,ratswereadministeredwithchronic
antihypertensivetherapyorvehicle(notreatment)viatheirdrinkingwaterforaperiodof2
weeks.Theanti-hypertensiveagentsusedwereperindopril(1mg/kg/day),orrilmenidine
(2mg/kg/day),whichwehaveshowntohaveantihypertensiveproperties[20,23–25].We
determinedtheconcentrationbymeasuringthewaterintakeoftheratsforatleast48hours
andadjustingindividualwaterintakeandbodyweightoftheratsonaweeklybasis.Afurther
3-daytelemetrymonitoringperiodwasperformedfromDay4–7andDay12–14ofthedrug
PLOSONE|DOI:10.1371/journal.pone.0168425 December21,2016 3/19
SympatheticNervousSystemContributiontoArousalBloodPressureSurge
treatmentperiod(i.e.day5,6,7,12,13,14).Theaverageoverthesedayswasusedtodetermine
theeffectofeachtreatment.Tenratsreceivedasingletreatment(vehiclen=4,perindopril
n=3,rilmenidinen=3),tworatsreceived2treatments(vehicleandrilmenidine),while5
receivedallthreetreatmentswithafourteen-dayrecoveryperiodallowedbetweentreatments.
Thustherewere17ratswiththetotalnumberoftreatmentsbeing11,8and10forvehicle,
perindoprilandrilmenidine,respectively.Themixtureoftreatmentswasdesignedtoincludea
degreeofwithinanimalconsistencyacrossthegroupswithsufficientnaïveanimalstoensure
thatthetreatmentswereonlysomewhatoverlapping.Thedesignminimisedtheuseofanimals
whichisdesirablefromananimalethicspointofview.
Dataanalysis
DataforBP,HRandbehavioralactivitywereaveragedover10minuteperiodsusingaspecially
writtenanalysisandfittingprogram(CIRCADversion2003).Thedatacorrespondingtoeach
heartbeatwasinitiallyfilteredinordertorejectindividualdatapointsthatexceeded2.5times
arunningstandarddeviation.Thevalueswerethenbinnedintohourlyvaluesandfittedtoa
doublelogisticcurvewiththeequation:
P P
y¼P (cid:0) P þ 2 þ 2 ð1Þ
1 2 1þeP3(cid:3)ðx(cid:0) P4Þ 1þeP5(cid:3)ðx(cid:0) P6Þ
whereP representedthe’high’plateau,i.e.dataobtainedduringthedarkperiodandP repre-
1 2
sentedthedatarange,i.e.thedifferencebetweenthecalculated’high’plateauand’low’plateau.
ThedifferencebetweenP andP thengivesthe’low’plateauwhichindicatesdataobtained
1 2
predominantlyduringthelightperiod.Thetransitionbetween’low’and’high’plateauswas
determinedasP andP whicharerateofchangecoefficients.Thesereflectthesteepnessof
3 5
transitionfrom’high’to’low’and’low’to’high’plateaus,respectively,areindependentofthe
absolutelevelandcanbeusedtocomparedifferentphysiologicalparameters.P andP arethe
4 6
calculatedtimesatwhichtherespectivetransitionsreach50%.Themethoddoesnotassume
thattheplateauduringthedayornightisofanyparticularlengthandsotheycanbequite
short.Inthiswaythemodelcancopewithdatathatslowlyincreaseordecreaseoverseveral
hoursorwithdatathatshowaparticularlyshortpeakortroughwhichoccasionallyoccursin
someanimals.
TheprogramCIRCADusesaniterativeleast-squaresfittingprocedurebaseduponthe
Marquardtalgorithm[26]withstartingparametersforthedoublelogisticfitobtainedfrom
aninitialCosinorfittingprocedure.Somelimitationsweresetonparameterstoensuresen-
siblefinalestimateswereobtained.TheconstraintsforP andP2wereestablishedwithan
1
initial’squarewave’fitofthedatawhichdeterminedthemeanvaluesandstandarddevia-
tion(SD)forthe’lighttime’andthe’darktime’.The‘darktime’mean+2SDwastakenasy
max,andthe’lighttime’mean—2SDwastakenasymin.TheconstraintsforP1andthe
rangeP2weresuchthatthefollowingwastruei)ymin<=P1<ymaxii)P2>0andiii)
ymin<=(P1+P2)<ymax.ConstraintsforthecurvatureparametersP3andP5weresuch
thattheentiretransitionbetweenplateauswasatleast30minuteswhichmeantthatatleast
10-minutedatapointsmustbeincludedintheslopeportion.Afterfittingthe6parameters
oftheequation,derivedvariableswereobtainedsuchasslopewhichistherateoftransition
betweenthe’high’and’low’plateau.Maximumslopeisdefinedas(P2(cid:3)P3)/4forthetransi-
tionbetweenthe’high’(darkperiod)plateauandthe’low’(lightperiod)plateauandas
(P2(cid:3)P5)/4forthetransitionbetweenthe’low’and’high’plateau.Theformulafortheslope
isthesameasthatusedforsinglelogisticcurvefittingasappliedtobaroreflexfunction
curves[27].
PLOSONE|DOI:10.1371/journal.pone.0168425 December21,2016 4/19
SympatheticNervousSystemContributiontoArousalBloodPressureSurge
Thepowerofthetransitionwascalculatedasthefirstderivativeofthelogisticcurvemulti-
pliedbytheamplitudewhichisthedaynightdifferencebetweenplateaus(Eq2)
P2(cid:2)P2(cid:2)P5(cid:2)eP5ðP6(cid:0) xÞ
^y ¼ ð2Þ
ð1þeP5ðP6(cid:0) xÞÞ2
ThemaximumpoweratthemidpointpeakofthecurvecanbecalculatedasinEq3
P2(cid:2)P2(cid:2)P5
^y ¼ ð3Þ
4
Cardiovascularvariabilityandcardiacbaroreceptorsensitivity
Attheendof2weeksoftreatment,beat-to-beatdatafrom72-hourrecordingswereanalyzed
separatelytocalculatepowerspectrausingaprogramwritteninLabview[28].Theauto-and
cross-powerspectrawerecalculatedformultipleoverlapping(by50%)segmentsofMAPand
HRusingaFastFouriertransformasadaptedforconsciousrats[29].Thecardiacbaroreflex
sensitivitywasestimatedastheaveragevalueofthetransfergaininthemidfrequencyband
(MF)between0.25and0.6Hz[29,30].Baroreflexslopewasconsideredsignificantifthe
coherencebetweenMAPandHRacrossseveraloverlappingsegmentsintheanalyzedfre-
quencybandwas>0.4.Coherenceisthefrequencydomainequivalentofthecorrelationcoef-
ficientandreflectshowmuchoftheHRoscillationisduetoBP.Datafromthelight(inactive)
periodanddark(active)periodwithlowlocomotoractivitywerechosen(4spectra)fromthe
72-hourrecordings,minimizingtheinfluenceofphysicalactivity.Lowfrequency(LF)MAP
powerwasdeterminedbetween0.05and0.25Hzandhighfrequency(HF)between0.6and
1.0Hz.
Statisticalanalysis
Dataarepresentedasmean±standarderrorofthemean(S.E.M.)ofthebetween-animalvari-
ation.AllparametersexceptP3andP5satisfiedthenormaldistribution.Forthelatterweper-
formedalogtransformwhichnormalisedthedistributioninallcases.Differencesbetween
curveparametersforagivenphysiologicalmeasureorbetweenphysiologicalmeasuresfora
givencurveparameterwerecomparedwithingroupswithatwo-wayrepeatedmeasuresanaly-
sisofvariance(ANOVA)followedbyposthocpairedttestsandbetweengroupcomparisons
weremadeusingaone-wayANOVA.FamilywiseerrorwasaccountedforbytheBonferroni
procedure.DifferenceswereconsideredsignificantwhenP<0.05.
Results
Effectofrilmenidineafter2weeksoftreatment
ThemaineffectofchronictreatmentwithrilmenidinewastoreduceSAP,MAPandDAPby
-7.5±1.9mmHg,-7.2±1.7mmHgand-6.2±1.7mmHgrespectively(P<0.01,Table1,“Fig
1”).TheeffectonSAPwas2foldgreaterduringtheactiveperiodatnightcomparedtotheday
period(-9.3±2.3mmHgvs-3.9±1.8mmHg,P=0.003,Table1,“Fig1”).Therewasasmaller
effectofrilmenidineonDAPbutwhichwassimilarduringthedayandnight(-5.1±1.6mmHg
and-5.1±2.0mmHgrespectively,P=0.01,Table1,“Fig1”).Therewasareductionintheday
nightdifferenceinSAPcausedbyrilmenidineof-5.4±1.7mmHg(P=0.002,Table1,“Fig1”).
ChronictreatmentwithrilmenidinewaslesseffectiveatloweringSAPinSHR(P=0.04)butno
differenceintheDAPwasobservedbetweenperindoprilandrilmenidinetreatedSHR
(P=0.09).ChronictreatmentwithrilmenidinereducedHRandactivityduringtheactive
PLOSONE|DOI:10.1371/journal.pone.0168425 December21,2016 5/19
SympatheticNervousSystemContributiontoArousalBloodPressureSurge
Table1. Averageresultsfromthecircadiananalysisof24-hourbloodpressuremeasurementsinratstreatedwithrilmenidine.
Total DiurnalRange DayPlateau NightPlateau
Variable Control Ril P Control Ril P Control Ril P Control Ril P
SAP(mmHg) Mean 153.9 146.3 *** 15.1 9.7 ** 146.8 142.9 NS 161.9 152.6 **
SEM 2.35 2.60 1.38 0.98 2.20 2.49 2.61 3.09
MAP(mmHg) Mean 132.2 125.0 *** 10.8 8.6 NS 127.1 121.6 ** 137.9 130.3 **
SEM 1.96 2.38 0.83 0.92 1.88 2.38 1.92 2.61
DAP(mmHg) Mean 111.9 105.6 ** 8.2 8.2 NS 107.8 102.7 ** 116.0 110.9 *
SEM 1.73 2.31 0.48 1.07 1.67 2.33 1.68 2.36
HR(b/min) Mean 283.6 270.4 ** 79.7 68.8 * 247.1 240.6 NS 326.8 309.5 **
SEM 3.2 3.3 3.8 2.9 3.6 2.9 4.2 4.6
Activity(Units) Mean 43.7 37.4 ** 48.9 28.0 ** 22.2 23.4 NS 71.2 51.4 **
SEM 3.7 4.2 6.9 2.3 3.5 3.5 7.2 4.4
DarktoLight LighttoDark DarktoLight LighttoDark
Rate Rate Power Power
Control Ril P Control Ril P Control Ril P Control Ril P
SAP Mean -2.55 -2.80 NS 2.72 1.34 ** -45.94 -38.17 NS 50.54 17.08 **
SEM 0.39 0.40 0.30 0.23 8.10 8.04 8.61 3.63
MAP Mean -2.30 -2.18 NS 2.23 1.18 NS -29.93 -25.01 NS 30.40 13.94 NS
SEM 0.31 0.31 0.31 0.24 4.77 4.77 5.90 4.06
DAP Mean -1.86 -1.86 NS 1.76 1.24 NS -18.57 -20.32 NS 18.17 15.63 NS
SEM 0.24 0.29 0.26 0.23 3.03 3.71 3.39 5.57
HR Mean -14.55 -7.62 NS 27.17 16.63 NS -1164 -585 * 2388 1261 NS
SEM 3.72 1.21 5.98 1.89 229 78 470 122
Activity Mean -6.21 -3.11 NS 10.84 6.74 ** -4.24 -1.04 NS 6.78 2.45 **
SEM 1.32 0.63 0.86 0.80 1.49 0.22 1.36 0.41
AbsolutevaluesandSEMshownineachcolumnoverthe24-hourperiod.
Significance:
*P<0.05,
**P<0.01,
***P<0.001,NSP>0.05.
TheunitsofratearemmHg/hour,b/min/hourorunits/hour.TheunitsforpoweraremmHg2/hour,b/min2/hourorunits2/hour.Abbreviations:RilRilmenidine,
SAPsystolicarterialpressure,MAPmeanarterialpressure,DAPdiastolicarterialpressure,HRheartrate.
doi:10.1371/journal.pone.0168425.t001
periodby5%and28%respectively(P<0.01)butwedidnotdetectanyeffectofrilmenidineon
thesevariablesduringtheinactiveperiod(Table1,“Fig1”).
RilmenidinetreatmentreducedthepowerofthetransitionofSAPfromlighttodark
from50.5to17mmHg2/hwhichisa~65%reduction(P<0.01)andtherateby~50%(from
2.7to1.3mmHg/h,P<0.01)buthadlittleeffectontherateofchangeorpowerofSAPdur-
ingthetransitionfromdarktolight(Table1,“Fig1”).BycontrastDAPduringeithertransi-
tionwasunaffectedbyrilmenidine.Further,therateofchangeandpowerofthetransitions
ofHRorlocomotoractivityweredifferentiallyaffectedbyrilmenidine.Therateandpower
oftheHRandlocomotorreductionduringthetransitionfromdarktolightandrateand
poweroftheriseduringthearousaltransitionalltendedtobelessinthepresenceofrilme-
nidinebutonlytheHRreductionandtheactivitypowerreachedstatisticalsignificance
(Table1,“Fig1”).
PLOSONE|DOI:10.1371/journal.pone.0168425 December21,2016 6/19
SympatheticNervousSystemContributiontoArousalBloodPressureSurge
Fig1.Averagedoublelogisticfittedcurvesshowingthecircadianvariationofsystolicanddiastolic
bloodpressure,heartrateandbehaviouralactivityovera24-hourperiodfrom10SHRbefore(open
circles)andafter(filledcircles)treatmentwithrilmenidinefor2weeks.Curveswereconstructedusing
theaverageparametersofdouble-logisticcurvefittingprocedure.ErrorbarsindicateSEMofhourlyaverages.
doi:10.1371/journal.pone.0168425.g001
Effectofperindoprilafter2weeksoftreatment
Chronicadministrationofperindoprilfor2weeksinthedrinkingwaterledtoamarkedreduc-
tioninSAP,MAPandDAPwhichaveragedover24hoursas21±3mmHg,19±3mmHgand
PLOSONE|DOI:10.1371/journal.pone.0168425 December21,2016 7/19
SympatheticNervousSystemContributiontoArousalBloodPressureSurge
Fig2.Averagedoublelogisticfittedcurvesshowingthecircadianvariationofsystolicanddiastolic
bloodpressure,heartrateandbehaviouralactivityovera24-hourperiodfrom8SHRbefore(open
circles)andafter(filledcircles)treatmentwithperindoprilfor2weeks.Curveswereconstructedusing
theaverageparametersofdouble-logisticcurvefittingprocedure.ErrorbarsindicateSEMofhourlyaverages.
doi:10.1371/journal.pone.0168425.g002
16±3mmHgrespectively(Table1,P<0.001,“Fig2”).Furthermore,thereductioninBPwas
similarduringboththenightanddayperiod(MAPwas18±4mmHglowerinthedayand
19±3mmHgduringthenight,P<0.001).Thustherewasnochangetothediurnaldaynight
difference(-1±3mmHg,Table2,“Fig2”).Locomotoractivitywasslightlyreducedby
PLOSONE|DOI:10.1371/journal.pone.0168425 December21,2016 8/19
SympatheticNervousSystemContributiontoArousalBloodPressureSurge
Table2. Averageresultsfromthecircadiananalysisof24-hourbloodpressuremeasurementsinratstreatedwithPerindopril.
Total DiurnalRange DayPlateau NightPlateau
Variable Control Perind P Control Perind P Control Perind P Control Perind P
SAP(mmHg) Mean 154.2 133.1 *** 13.8 10.9 NS 146.6 127.8 *** 160.4 138.7 ***
SEM 3.67 4.94 0.81 1.34 3.26 4.85 3.56 5.06
MAP(mmHg) Mean 134.1 115.3 *** 10.6 9.2 NS 128.7 110.8 *** 139.2 120.0 ***
SEM 2.87 4.68 0.70 1.08 2.86 4.70 2.91 4.67
DAP(mmHg) Mean 115.1 99.6 *** 9.0 8.4 NS 110.9 95.3 *** 119.8 103.7 ***
SEM 3.00 4.99 0.79 1.05 2.84 5.08 3.22 4.85
HR(b/min) Mean 282.7 294.1 * 76.0 82.5 NS 248.6 258.3 NS 324.6 340.8 NS
SEM 5.9 5.7 3.8 4.4 6.2 5.1 5.2 5.5
Activity(Units) Mean 47.5 42.5 * 49.4 41.4 NS 24.6 23.4 NS 73.9 64.7 *
SEM 5.4 4.3 3.8 3.1 4.8 4.4 7.1 5.8
DarktoLight LighttoDark DarktoLight LighttoDark
Rate Rate Power Power
Control Perind P Control Perind P Control Perind P Control Perind P
SAP Mean -2.82 -1.73 * 2.87 2.54 NS -45.29 -23.20 * 47.27 41.46 NS
SEM 0.42 0.31 0.36 0.51 7.67 5.99 6.78 11.17
MAP Mean -2.28 -1.56 NS 2.47 2.03 NS -29.91 -17.37 NS 31.20 27.78 NS
SEM 0.39 0.27 0.30 0.44 6.37 4.57 4.70 8.51
DAP Mean -1.88 -1.54 NS 1.95 1.50 NS -22.39 -18.04 NS 21.84 16.79 NS
SEM 0.37 0.33 0.27 0.30 5.79 5.43 4.07 4.68
HR Mean -23.57 -22.68 NS 31.18 52.07 NS -1804 -1989 NS 2613 4811 NS
SEM 5.87 5.78 7.19 12.18 406 462 574 1142
Activity Mean -6.81 -7.44 NS 12.38 9.13 NS -3.67 -3.90 NS 7.68 4.60 *
SEM 1.06 1.49 1.31 1.25 0.54 0.91 1.29 0.81
AbsolutevaluesandSEMshownineachcolumnoverthe24-hourperiod.Significance:
*P<0.05,
**P<0.01,
***P<0.001,
NSP>0.05.TheunitsofratearemmHg/hour,b/min/hourorunits/hour.TheunitsforpoweraremmHg2/hour,b/min2/hourorunits2/hour.Abbreviations:
PerindPerindopril,SAPsystolicarterialpressure,MAPmeanarterialpressure,DAPdiastolicarterialpressure,HRheartrate.
doi:10.1371/journal.pone.0168425.t002
perindoprilbutonlyduringthedarkperiod(-11%,P=0.005,Table2,“Fig2”).Perindopril
treatedSHRhadslightlyhigherHRthanatbaseline(Table2,“Fig2”).Perindopriltreatment
hadnoeffectonBP,HRorlocomotoractivityduringthetransitionperiodsofdaytonightor
nighttodaysincetherateofchangeandthepowerofthechangewassimilarinthecontroland
treatmentphases(Table2,“Fig2”).
Effectof2weeksofvehicletreatment
VehicletreatedSHRattheendof2weekshadslightlyhigherSAP,MAPandDAPthanatcon-
trol(forMAP+5.1±2.0mmHg,P<0.05,Table3,“Fig3”).Thiseffectwasevidentduringboth
thedayandnightperiods.BycontrastHRandactivitywerecloselysimilarbeforeandafter
treatment(Table3,“Fig3”).Therewerenodifferencesdetectedintheday-nightrange,rateof
transitionorpowerforanyvariable(Table3,“Fig3”).
PLOSONE|DOI:10.1371/journal.pone.0168425 December21,2016 9/19
SympatheticNervousSystemContributiontoArousalBloodPressureSurge
Table3. Averageresultsfromthecircadiananalysisof24-hourbloodpressuremeasurementsinratstreatedwithVehicle.
Total DiurnalRange DayPlateau NightPlateau
Variable Control Vehicle P Control Vehicle P Control Vehicle P Control Vehicle P
SAP(mmHg) Mean 150.4 155.2 * 15.9 15.9 NS 142.8 148.0 * 158.7 163.9 *
SEM 2.40 2.61 1.38 1.60 2.58 2.70 2.74 2.80
MAP(mmHg) Mean 130.8 135.9 * 10.9 12.2 NS 125.6 130.1 * 136.5 142.3 *
SEM 2.20 2.11 0.73 1.43 2.37 2.24 2.32 2.18
DAP(mmHg) Mean 112.2 117.6 * 8.9 10.5 NS 108.1 112.6 * 117.0 123.0 *
SEM 2.68 2.28 0.71 1.08 2.68 2.28 2.77 2.41
HR(b/min) Mean 287.5 285.6 NS 74.8 75.0 NS 252.9 250.1 NS 327.7 325.1 NS
SEM 3.1 2.6 2.5 2.9 3.4 2.5 3.3 3.1
Activity(Units) Mean 50.5 51.4 NS 43.7 42.6 NS 27.5 31.2 * 71.1 73.8 NS
SEM 4.2 4.3 2.8 6.0 4.2 4.7 5.2 6.5
DarktoLight LighttoDark DarktoLight LighttoDark
Rate Rate Power Power
Control Vehicle P Control Vehicle P Control Vehicle P Control Vehicle P
SAP Mean -2.35 -2.51 NS 2.12 2.36 NS -45.04 -48.13 NS 42.06 48.45 NS
SEM 0.32 0.34 0.22 0.26 7.51 7.66 6.67 9.68
MAP Mean -2.76 -2.75 NS 1.94 2.16 NS -38.50 -40.28 NS 25.67 33.74 NS
SEM 0.37 0.30 0.22 0.25 6.85 6.30 3.55 7.16
DAP Mean -2.35 -2.39 NS 1.64 1.70 NS -27.86 -31.68 NS 18.23 23.03 NS
SEM 0.30 0.27 0.20 0.20 5.36 5.28 2.69 4.28
HR Mean -14.69 -15.11 NS 40.75 36.51 NS -1162 -1191 NS 3431 2993 NS
SEM 2.79 2.57 7.31 5.84 191 169 642 440
Activity Mean -6.18 -5.95 NS 10.55 9.59 NS -3.01 -3.12 NS 5.71 5.39 NS
SEM 0.91 0.75 1.07 0.84 0.46 0.87 0.83 1.22
AbsolutevaluesandSEMshownineachcolumnoverthe24-hourperiod.Significance:
*P<0.05,
**P<0.01,
***P<0.001,
NSP>0.05.TheunitsofratearemmHg/hour,b/min/hourorunits/hour.TheunitsforpoweraremmHg2/hour,b/min2/hourorunits2/hour.Abbreviations:SAP
systolicarterialpressure,MAPmeanarterialpressure,DAPdiastolicarterialpressure,HRheartrate.
doi:10.1371/journal.pone.0168425.t003
EffectoftreatmentsonarousalsurgepowerfunctioncurvesofSAP
Duringtheonsetofthedarkactiveperiod,thepowerofthesurgecanbeplottedusingthe
averagecalculatedvariablesforthederivativeofthelogisticequation.Thepeakofthepower
surgeoccurs2–3hoursaftertheonsetofthedark(lightsoff)(“Fig4”).Themaximumandthe
timingofthepeakwerenotaffectedbyperindoprilorbyvehicle.However,rilmenidine
markedlyattenuatedthemaximumofthecurvesuchthatitoccurred2–3hoursbeforethe
onsetofdarkness(“Fig4”).
Bloodpressurevariability
Duringthedark(active)period,MFMAPpowerwas105%greaterinvehicletreatedSHR
(P<0.001)and87%greaterinperindopriltreatedSHR(P<0.001)thanthatobservedduring
thedayinactiveperiod.However,wedidnotobserveadaynightdifferenceinrilmenidine
treatedSHR(+29%,P=0.7,“Fig5”).ThuswhilenotreatmentsaffectedtheMFMAPpower
inthedayinactiveperiod,rilmenidinereducedthepowertosimilarlevelsasthatobserved
PLOSONE|DOI:10.1371/journal.pone.0168425 December21,2016 10/19