Protracted hypomobility in the absence of

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Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/

Title

Protracted hypomobility in the absence of

trigeminal sensitization after cortical spreading depolarization: Relevance to migraine postdrome

Author(s) Alternative

Shibata, M; Kitagawa, S; Tang, C; Unekawa, M;

Kayama, Y; Shimizu, T; Nakahara, J; Suzuki, N Journal Neuroscience research, 172(): 80‑86

URL http://hdl.handle.net/10130/5618

Right

© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Description

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ContentslistsavailableatScienceDirect

Neuroscience Research

j o ur na l h o me pa g e :w w w . e l s e v i e r . c o m / l o c a t e / n e u r e s

Protracted hypomobility in the absence of trigeminal sensitization after cortical spreading depolarization: Relevance to migraine postdrome

Mamoru Shibata

^a,b,∗,1

, Satoshi Kitagawa

^a

, Chunghua Tang

^a,c

, Miyuki Unekawa

^a

, Yohei Kayama

^a

, Toshihiko Shimizu

^a

, Jin Nakahara

^a

, Norihiro Suzuki

^a,d

aDepartmentofNeurology,KeioUniversitySchoolofMedicine,Tokyo,160-8582,Japan

bDepartmentofNeurology,TokyoDentalCollegeIchikawaGeneralHospital,Chiba,272-0513,Japan

cDepartmentofNeurologyandCentreforClinicalNeuroscience,DapingHospital,ThirdMilitaryMedicalUniversity,Chongqing,400042,China

dDepartmentofNeurology,ShonanKeiikuHospital,Kanagawa,252-0816,Japan

a rt i c l e i nf o

Articlehistory:

Received20January2021

Receivedinrevisedform5March2021 Accepted28March2021

Availableonline2April2021

Keywords:

Migraine

Corticalspreadingdepolarization Photophobia

Physicalactivity Migrainepostdrome Neurotropin^®

a b s t ra c t

Migrainesufferersoftenexhibitphotophobiaandphysicalhypoactivityinthepostdromeandinterictal periods,forwhichnoeffectivetherapycurrentlyexists.Corticalspreadingdepolarization(CSD)isa neuralphenomenonunderlyingmigraineaura.WepreviouslyreportedthatCSDinducedtrigeminal sensitization,photophobia,andhypomobilityat24hinmice.Here,weexaminedtheeffectsofCSD inductiononlightsensitivityandphysicalactivityinmiceat48hand72h.Trigeminalsensitizationwas absentatbothtimepoints.CSD-subjectedmiceexhibitedsigniﬁcantlylessambulatorytimeinbothlight (P=0.0074,theBonferronitest)anddark(P=0.0354,theBonferronitest)zonesthansham-operatedmice at72h.CSD-subjectedmicealsoexhibitedasigniﬁcantlyshorterambulatorydistanceinthelightzoneat 72hthansham-operatedmice(P=0.0151,theBonferronitest).Neurotropin^®isusedforthemanagement ofchronicpaindisorders,mainlyinAsiancountries.TheCSD-inducedreductionsinambulatorytimeand distanceinthelightzoneat72hwerereversedbyNeurotropin^®at0.27NU/kg.Ourexperimentalmodel seemstorecapitulatemigraine-associatedclinicalfeaturesobservedinthepostdromeandinterictal periods.Moreover,Neurotropin^®maybeeffectiveinamelioratingpostdromal/interictalhypoactivity, especiallyinalightenvironment.

1. Introduction

Migraine is a debilitating chronic neurological disorder characterized by recurrent headache attacks accompanied by nausea/vomiting and heightenedsensitivity tolight and sound (HeadacheClassiﬁcationCommitteeoftheInternationalHeadache Society,2018).Atypicalmigraineheadacheisunilateral,pulsat- ing,andaggravatedbyphysicalactivity(HeadacheClassiﬁcation CommitteeoftheInternationalHeadacheSociety,2018).Migraine, thesecondleadingcauseofyearslivedwithdisability(GBD2016

Abbreviations: CI,Conﬁdenceintervals;CSD,Corticalspreadingdepolarization;

FWHM,Fullwidthathalfmaximum;NF-␬B,Nuclearfactor-kappaB.

∗Correspondingauthorat:DepartmentofNeurology,KeioUniversitySchoolof Medicine,35Shinanomachi,Shinjuku-ku,Tokyo,160-8582,Japan.

E-mailaddresses:[email protected],[email protected](M.Shibata).

1 Presentaddress:DepartmentofNeurology,TokyoDentalCollegeIchikawaGen- eralHospital,5-11-13Sugano,Ichikawa,Chiba272-8513,Japan.

DiseaseandInjuryIncidenceandPrevalenceCollaborators.,2017), not only interferes withthe daily lives of individuals, but also posesanenormouseconomicburdenonsociety(Fosteretal.,2020;

Takeshimaetal.,2019).Migraine hasdistinct phases,including premonitory,aura, headache,postdrome,andinterictal (Dodick, 2018b).Although peoplewithmigraine suffer most duringthe headachephase,neurologicalsymptomsareknowntopersistin thepostdrome andinterictal phases(deTommaso etal., 2014;

May,2017).Ithasbeenshownthatabnormalsensitivitytolight (photophobia)andocularallodyniaarepresentinbothictaland interictalphasesof migraine(Chu etal.,2011; McAdamsetal., 2020;Mullenersetal.,2001;Perenboometal.,2018).Thesesymp- tomscanmakeworkinginalightenvironmentmoredifﬁcult.For instance,affectedindividualsmayexperiencedifﬁcultyworkingon computerscreensforaprolongedtime,whichisaconsiderabledis- advantagegiventhecontemporaryworkstyle.Moreover,recent datahavedemonstratedthatpatientswithmigrainehavereduced physicalactivityonbothnon-headachedaysandheadachedays https://doi.org/10.1016/j.neures.2021.03.010

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M.Shibataetal. NeuroscienceResearch172(2021)80–86

(Bondetal.,2015;Rogersetal.,2020).Asthenia,weakness,tiredness,andlightsensitivityhavebeenidentiﬁedasmajorsymptoms experienced during themigraine postdrome (Blau, 1982; Gifﬁn et al.,2016; Kelman,2006;Ng-Maket al.,2011; Quintelaetal., 2006).

Thetrigeminovascularsystemisthoughttoplayapivotalrole inmigrainepathogenesis(Ashinaetal.,2019).Nosedaetal.(2011) clariﬁedthatthird-ordertrigeminovascularthalamicneuronssend heavyprojectionstothemotorand visualcortices,thusprovid- ing a neuroanatomical basis for theemergence of photophobia and hypoactivityin migraine. Cortical spreading depolarization (CSD),aneurophysiologicalcorrelateofmigraineaura(Charlesand Baca, 2013; Pietrobonand Moskowitz,2014), is knowntoacti- vate thetrigeminovascularsystem(Iwashitaetal.,2013;Zhang etal.,2011,2010).WepreviouslydemonstratedthatCSDcaused trigeminal sensitization, photophobia, and hypoactivityin both light anddarkzonesat24hinmice,allofwhichwereamelio- ratedbyanti-migraineagents,sumatriptanandolcegepant(Tang etal.,2020).However,itremainsunclearwhetherthesesymptoms persistbeyond24 hafterCSDinduction.Ifdetected,prolonged abnormalitiesoftrigeminalsensation,lightsensitivity,andphysical activitywouldprovidea diseasemodelforstudyingthemecha- nismsunderlyingpostdromeandinterictalphasesofmigraine.Our behavioralassayallowsustoexploretheeffectofCSDonmobility beyond24h.

Neurotropin^® is a non-protein fraction extracted from the inflamedskinofrabbitsafterinoculationwithvacciniavirus,and hasbeentraditionallyusedforthetreatmentofvariouschronic painconditions,mainlyinAsiancountries.Clinicalevidenceforthe efficacyofNeurotropin^®inalleviatingheadachedisorderisscarce, althoughithasbeenreportedthattreatmentwithNeurotropin^® relieved nummular headache (Danno et al., 2013). Given that Neurotropin^® contains a multitude of bioactive substances, it exertsvariousactions,rangingfromneurotrophiceffects(Fukuda etal.,2010,2015;Nakajoetal.,2015)toinflammatorymodula- tion(Fukudaetal.,2020;Katoetal.,1991;Nishimotoetal.,2016;

Yoshiietal.,1987).Numerouscomplexpathwaysarethoughttobe involvedintheanalgesiceffectofNeurotropin^®.

In the present study,we examined CSD-inducedchanges in trigeminal sensation and physical activity at 48 and 72 h. We alsoexploredtheeffectofNeurotropin^®administrationonCSD- inducedphysicalactivity.

2. Materialsandmethods 2.1. Animals

ThepresentstudywasapprovedbytheLaboratoryAnimalCare andUseCommitteeofKeioUniversity(approvalno.14,084).Male C57BL/6miceaged8–12weekswereobtainedfromCLEAJapanInc.

(Fujinomiya,Japan).Atotalof55micewereusedinthepresent study.Theywerekeptinanambientspeciﬁc-pathogen-freecondi- tionwitha12-hlight/darkcycleandhadfreeaccesstofoodand water. Allexperimentswere performedinaccordance withthe NationalInstituteofHealthGuidefortheCareandUseofLaboratory Animals(NIHPublicationsNo.80-23)revisedin1996.

2.2. Drugadministration

Neurotropin^® solution(20 NU/mL;Nippon Zoki Pharmaceu- tical Co., Ltd., Osaka, Japan) was dissolved in normal saline.

Neurotropin^®wasslowlyadministeredat0.27NU/kgor0.81NU/kg in200␮Lsolutionusingafeedingneedle(18060−20,FineScience Tools,FosterCity,USA).Thesedosesarecomparabletothoseused fortreatingneuropathicpain.Thesamevolumeofnormalsaline

wasadministeredasavehicle.Drugadministrationwascarriedout 30minbeforeCSDinduction.Therewerefourdifferentexperimen- talgroupsinthepresentstudy,asfollows:theSham-Vehiclegroup, theCSD-Vehiclegroup,theCSD-NT0.27(Neurotropin^®0.27NU/kg) group,andtheCSD-NT0.81(Neurotropin^®0.81NU/kg)group.

2.3. CSDinduction

Underisoﬂuraneanesthesia(1–2%),themouseheadwasﬁxed in a stereotaxic apparatus. Non-invasive monitoring of systolic bloodpressureandheartratewasperformedusinganon-invasive bloodpressuremonitor(MK-2000ST;MuromachiKikaiCo.,Ltd., Tokyo,Japan).Bodytemperaturewaskeptataround37^◦Cusing athermistor-regulatedheatingpad(BWT-100;BioresearchCenter Co.,Ltd.,Nagoya,Japan).

For electrophysiological recordings, three burr-holes were drilledintotheleftpartoftheskull.Theposteriorholeatthecoor- dinatesof5mmposteriorand2mmlateraltothebregmawas usedforKCladministration.Theparietalhole(2mmlateraland 2mmcaudaltothebregma)andthefrontalhole(2mmlateral and2mmrostraltothebregma)weredrilledtoinstalltherecord- ingelectrodes.TwoAgA/gClDCelectrodes(tipdiameter=200␮m, EEG-5002Ag;BioresearchCenterCo.,Ltd.)wereinstalledonthe dura at theparietal (proximal) and frontal(distal) holes. A DC potentialwasappliedat1–100Hzanddigitizedat1 kHzusing anextracellularamplifier(Model4002andEX1;DaganCo.,Min- neapolis,MN,USA).ContinuousrecordingsoftheDCpotentialwere stored on a multi-channel recorder(PowerLab 8/30; ADInstru- ments,Ltd.,Sydney,Australia).Afterconfirmingthatallparameters werestableforatleast10min,CSDwasinducedbyapplyingKCl solution(1.0mol/L,5␮L)ontothedura.TheinductionofCSDwas verifiedbytheappearanceofadistinctDCpotentialdeflection.CSD wasinducedfivetimesafterwashingthecorticalsurfacewithnor- malsaline.Afterthesurgery,allelectrodeswereremoved,andthe craniotomieswereclosed.Insham-operatedmice,allsurgicalpro- cedureswereperformedexceptforthechemicalstimulationwith KClsolutionandDC electrodeinsertion.Sham-treatedmiceand CSD-subjectedmicewereanesthetizedforequivalentperiodsof time.Inthepresentpaper,werefertotheCSDoperationandsham operationcollectivelyassurgery.

TheCSDpropagationvelocityandfullwidthathalfmaximum (FWHM)weremeasured.TheCSDpropagationvelocitywascalcu- latedonthebasisofthelatencyanddistancebetweentheproximal anddistalelectrodes.TheFWHMwasdeterminedfromthecurves recordedatthedistalelectrode.

2.4. Facialheatpainthresholdtemperaturemeasurement

Thedetailedprotocolforheatpainthresholdtemperaturehas beendescribedelsewhere(Kayamaetal.,2018).Brieﬂy,afteraccli- mationtotheexperimentalapparatusandfacialhairremoval,a pairofPeltiermodulebarswithasurfacetemperatureregulated between36^◦Cand56^◦Cwasappliedbilaterallytothelateralaspect oftheface.Thebarsurfacetemperaturewasgraduallyelevated from36^◦Cby 1^◦C/4 suntil facewithdrawal. Mousebehaviors weremonitoredusingavideorecorder(Panasonic,Kadoma,Japan).

Videoswereanalyzedbyanexaminerwhowasblindedtogroup allocationoftheanimals.Thelowesttemperatureatwhichamouse turneditshead away fromtheheating bars wasconsideredas theheatpainthresholdtemperature.Ineachsession,thethresh- oldtemperaturewasmeasuredﬁvetimes.Themeasurementwas carriedoutbeforesurgeryand48hand72haftersurgery.

Inourpreliminaryexperiments,thestandarddeviationofthe heatpainthresholdtemperatureofnormalmicewasfoundtobe 0.8–1.0^◦C.WhenthetypeIerrorrateandpowerwere5%and0.80, 81

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respectively,thesamplesizerequiredtodetecta0.5^◦Cdifference wascalculatedas40–60trialsineachgroup.

2.5. Mouselocomotionassayinthelightanddarkzones

Thedetailed protocolforthis assayhasbeendescribedelse- where(Tangetal.,2020).Briefly,mousebehaviorwascontinuously recordedinatestingchamber(27cmwide×27cmdeep×20.3 cmhigh)withthreesetsof16-beaminfraredarrays(twosetsof perpendicularbeamscrossedataheightof1.0cmtodetectmouse locationandlocomotion,andthethirdbeamcrossedthewidthof thechamberataheightof7.3cmtodetectverticalactivity;Med Associates,Fairfax,VT).Thetestingfieldwasequallydividedinto lightanddarkzonesusingadarkinsert(MedAssociates).Themice wereallowedtomovebetweenthetwozonesthroughanopening (5.2cm×6.8cm).Thelightintensitymeasuredataheightof2cmin thelightzonewas540lx,whereastheintensitiesmeasuredinthe darkzonewere380lximmediatelyinsidetheorifice,20lxatthe center,and5lxatthecorners.Mouseactivitywasanalyzedusinga computerequippedwithActivityMonitorv6.02(MedAssociates).

Mice were allowed to acclimatize to the test chamber, as describedpreviously(Tangetal.,2020).Theacclimationwascar- riedoutpriortothesurgery.At48and72haftertheCSDorsham operation, locomotoractivitywasrecordedfor30 minwiththe lightson.Thetotaltimespentinthelightﬁeldwasusedtoeval- uatelightaversion.Forallmiceexamined,behavioraltestingwas carriedoutbetween10:00and18:00inaquietroom.

2.6. Assessmentofmouselocomotoractivity

The effects of Neurotropin^® administration onmouse loco- motion in both compartments were evaluated using several movementparameters.Whenmicemovedoutofthe6.35cm×6.35 cmsquareboxaroundthemwithin0.5s,theirmovementswere definedasambulatory.Ambulatorydistance(cm)wasdefinedas thetotaldistancetravelledduringambulatorymovement.Ambu- latoryaveragevelocity(cm/s)wascalculatedastheambulatory distancedividedbyambulatory time,whichwasdefinedasthe timespentintheambulatorymovementstatus.Ourpreliminary experimentsrevealedthatthestandarddeviationofthetotaltime spentinthelightzoneofuntreatedcontrolmicewas120–150s.

WithatypeIerrorrateandpowerof5%and0.80,respectively,the samplesizerequiredtodetecta200sdifferencewascalculatedas 6–9subjectsineachgroup.

2.7. Statisticalanalyses

NumericaldatarelevanttophysiologicalandCSD-relatedelec- trophysiologicalparametersareexpressedasthemean±SD.The heatpainthresholdtemperaturesandlocomotiondataareshown using95%conﬁdenceintervals(CIs).Between-groupcomparisons of physiological parameters and CSD-related electrophysiological data were made using the Kruskal–Wallis test. A two-way repeated-measuresANOVAwasusedtoevaluatetheeffectsofCSD inductionandpharmacologicalinterventionsonthetemporaltra- jectoriesoftheheatpainthresholdtemperature,totaltimespentin thelightzone,ambulatorytime,anddistancetravelledineachzone.

Post-hocmultiplecomparisonswerecarriedoutusingtheBonfer- roni test. Meandifferencesareindicated by95 %CI.Data were analyzedusingGraphPadPrism8software(GraphPadSoftware, SanDiego,CA).

Table1

CSD-relatedelectrophysiologicalparameters.

CSD- Vehicle

CSD-NT 0.27

CSD-NT 0.81

P-value

Numberofanimals 8 8 8

Propagationvelocity (mm/min)

4.2±0.6 5.0±1.1 4.2±0.6 0.3901 FWHM(s) 41.3±3.3 42.8±10.1 45.8±2.9 0.4825

3. Results

3.1. Physiologicalparameters,bodyweight,andCSD-related electrophysiologicalparameters

Thetimelineofthepresent studyisshown inFig.1A.Cran- iotomies for CSD induction and installation of electrodes and experimentalprobesareshowninFig.1B.Arepresentativerecord- ing of CSD induction is illustrated in Fig. 1C, with concrete measurementsforCSDpropagationvelocity(Fig.1D)andFWHM (Fig.1E).Weobservednosigniﬁcantbetween-groupdifferencesin systolicbloodpressure,heartrate,orbodyweight(datanotshown).

TherewasnosigniﬁcanteffectofNeurotropin^®administrationon CSD-relatedelectrophysiologicalparameters(propagationvelocity andFWHM;Table1).

3.2. EffectsofCSDonfacialheatpainthresholdtemperatureat48 hand72hafterCSDinduction

Atwo-wayANOVAshowedthattherewasnosigniﬁcanteffectof timeorCSDinductiononthefacialheatpainthresholdtemperature at48and72haftersurgery(time:F_(2,228)=1.011,P=0.3655;CSD induction:F_(1,114)=0.05389,P=0.8168).Themeandifferencesof facialheatpainthresholdvs.thebaselinevaluewere-0.21^◦C(95

%CI:-0.74–0.32,P>0.9999)and-0.02^◦C(95%CI:-0.56–0.51,P>

0.9999)at48hand72h,respectively.

3.3. EffectsofCSDinductionandNeurotropin^®pretreatmenton thetotaltimespentinthelightzone

We exploredtheeffects ofCSD inductionand Neurotropin^® administrationonthetotaltimespentinthelightzoneat48hand 72haftersurgery.Atwo-wayANOVAdidnotdetectamaineffect ofobservationtimeonthetimespentinthelightzone(P=0.1486).

WhiletheeffectofCSDinductionandNeurotropin^®administration didnotreachsigniﬁcance(F_(1,28)=2.207,P=0.0787),therewasa trendtowardsashortertotaltimespentinthelightzoneinthe CSD-VehiclegroupascomparedwiththeSham-Vehiclegroupat 72h(meandifference:−327s[95%CI:-672.3–18.32],P=0.0732, theBonferronitest).Moreover,Neurotropin^®(0.27NU/kg)tended toreversetheCSD-induceddecreaseonthetimespentinthelight zone(meandifference:343.4s[95%CI:-1.941–688.7],P=0.0521, theBonferronitest).

3.4. EffectsofCSDandNeurotropin^®pretreatmenton ambulatorytimeinthelightanddarkzones

WethenanalyzedtheeffectsofCSDaloneandcombinedwith Neurotropin^® pretreatmentonambulatorytimeinthelightand darkzonesseparatelyat48hand72haftersurgery.Inthelight zone,atwo-wayANOVAdetectedasignificantmaineffectofCSD inductionand Neurotropin^® administration(F_(3,28) =5.254, P= 0.0053),whiletherewasnosignificantmaineffectofobservation time(F_(1,28)=0.372,P=0.5468)(Fig.2A).Therewasasignificant differenceinambulatorytimeinthelightzonebetweentheSham- VehiclegroupandtheCSD-Vehiclegroup(meandifference:-26.07

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Fig.1. ExperimenttimelineandmeasurementsforCSD-relatedelectrophysiologicalparameters.(A)Experimentaltimelineforthemeasurementoffacialheatpainthreshold temperatureandthelight/darkboxbehavioraltest.Facialheatpainthresholdmeasurementswereperformedimmediatelybeforeand48and72hafterCSDinduction (orangetriangles).Thelight/darkboxbehavioraltestingwasperformedat48and72hafterCSDinduction(greentriangles).Investigationaldrugs,includingthevehicle, wereadministered30minpriortoCSDinduction.

(B)CraniotomiesforCSDinductionandinstallationofexperimentalprobes.(C)ArepresentativerecordingofCSDinduction.(D)MeasurementofCSDpropagationvelocity.

(E)MeasurementofFWHM.

s[95% CI:-47.03–-5.109],P=0.0074, theBonferronitest)and betweentheCSD-VehiclegroupandtheCSD-NT0.27group(mean difference:29.65s[95%CI:8.69–50.61],P=0.0017,theBonfer- roni test)at72h(Fig.2A).A two-wayANOVAdidnot detecta signiﬁcantmaineffectoftimeinthedarkzone(F_(1,28)=0.09536, P=0.7598),whiletheeffectofCSDinductionwithNeurotropin^® administrationonambulatorytimeinthedarkzonenearlyreached signiﬁcance(F_(3,28)=2.717,P=0.0636)(Fig.2A).Therewasasignif- icantdifferenceinambulatorytimeinthedarkzonebetweenthe Sham-VehiclegroupandtheCSD-Vehiclegrouponly(meandifference:-22.55s[95%CI:-44.09–-1.007],P=0.0354,theBonferroni test)at72h(Fig.2A).

3.5. EffectsofCSDandNeurotropin^®pretreatmenton ambulatorydistanceinthelightanddarkzones

WealsoexaminedtheeffectsofCSDaloneandcombinedwith Neurotropin^®administrationonambulatorydistanceinthelight anddarkzonesat48hand72haftersurgery.Inthelightzone,a two-wayANOVArevealedasignificantmaineffectofCSDinduction andNeurotropin^®administrationonambulatorydistance(F_(3,28)= 5.138,P=0.0059),whiletherewasnosignificantmaineffectof observationtime(F_(1,28)=0.3289,P=0.5709)(Fig.2B).Therewas a significantdifferenceinambulatorydistanceinthelightzone betweentheSham-VehiclegroupandtheCSD-Vehiclegroup(mean difference:-933.5cm[95%CI:-1741–126.2],P=0.0151,theBon- ferronitest)andbetweentheCSD-VehiclegroupandtheCSD-NT 0.27group(meandifference:1142cm[95%CI:335.1–1950],P= 0.0017,theBonferronitest)at72h(Fig.2B).

Inthedarkzone,atwo-wayANOVAdidnotdetectanysigniﬁ- cantmaineffectsofobservationtime(F_(1,28)=0.4041,P=0.5301) orCSDinductionandNeurotropin^®pretreatment(F_(3,28)=2.469,P

=0.0827)onambulatorydistance(Fig.2B).TheCSD-NT0.27group tended toexhibit a greater ambulatory distance thanthe CSD-

Vehiclegroup(meandifference:750.7cm[95%CI:-5.714–1507], P=0.0528,theBonferronitest)at72h(Fig.2B).

3.6. Between-groupcomparisonofaverageambulatoryspeed

TheaverageambulatoryspeedwasunalteredbyCSD.Further- more,treatmentwithNeurotropin^® ateitherdosedidnotaffect theaverageambulatoryspeed(Fig.2C).

4. Discussion

Untreatedmigraineattacksmaypersistforupto72h(2018), which iswhy weexamined trigeminalsensitizationand physi- calactivityat48hand72hafterCSDinthepresentstudy.The mostnotableﬁndingwasthatCSDdeterioratedhypomotilityin thelightanddarkzones,evenaftertrigeminalsensitizationhad wornoff.Itisunlikelythatthisreducedlocomotoractivityresulted frommuscleweaknessandmotorparalysis,becausetheaverage ambulatoryspeedwasunaffectedbyCSD.Wepreviouslyreported thatthesameCSDinsultasthatusedinthepresentstudycaused trigeminalsensitization,photophobia,and hypomotilityat 24h ascomparedwithsham-operatedmice(Tangetal.,2020).How- ever,thepresentstudyrevealedthattherewerenobetween-group differencesat48 hinanyof theseparameters.At72 h, mouse ambulatorytimeexhibitedatendencytorecovercomparedtothat seenat48hinthesham-operatedmice,whereastheambulatory timeofCSD-affectedmicecontinuedtodeteriorate.Overall,CSD- inducedhypomobilityexhibitedadiphasicchange.Suchprolonged hypomotilitywasmoreevidentinthelightzone,asevidencedby signiﬁcantreductionsin both ambulatory time and ambulatory distance.Concomitantly,theCSD-affectedmicetendedtospend lesstimeinthelightzonethansham-operatedmice.Hence,itis suggestedthatlightaversionmayplayaroleinthehypomotility.

Ourpreviousdatademonstratedthattrigeminalthermalallody- niawaspresent at24 hin anidenticalCSDmodel(Tangetal., 83

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Fig.2.(A)Ambulatorytimeinthelightanddarkzonesineachgroup.Thegraphsshowtheambulatorytime(s).Redandbluebarsindicatelightanddarkdata,respectively.

Thedataarepresentedasmean±SD.Atwo-wayrepeated-measuresANOVAwasusedtoevaluatetheeffectsofCSDinduction,Neurotropin^®pretreatment,andobservation time.MultiplecomparisonswereperformedusingtheBonferronitest(*P<0.05,**P<0.01).N=8ineachgroup.

(B)Ambulatorydistanceinthelightanddarkzonesineachgroup.Thegraphsshowtheambulatorydistance(cm).Redandbluebarsindicatelightanddarkdata,respectively.

Thedataarepresentedasmean±SD.Atwo-wayrepeated-measuresANOVAwasusedtoevaluatetheeffectsofCSDinduction,Neurotropin^®pretreatment,andobservation time.MultiplecomparisonswereperformedusingtheBonferronitest(*P<0.05,**P<0.01).N=8ineachgroup.

(C)Averageambulatoryspeedineachgroup.Thegraphsshowtheaverageambulatoryspeed(cm/s).Thedataarepresentedasmean±SD.Atwo-wayrepeated-measures ANOVAwasusedtoevaluatetheeffectsofCSDinduction,Neurotropin^®pretreatment,andobservationtime.MultiplecomparisonswereperformedusingtheBonferroni test.N=8ineachgroup.

2020).Consistently,apreviousinvivostudyusingintravitalrecord- ingofelectricalactivityofthedura-innervatingtrigeminalganglion neuronsshowedthatCSDcausedmechanicalsensitization(Zhang etal.,2010).Furthermore,theﬁringrateofneuronsreceivingdural nociceptivesignalsinthetrigeminalnucleuscaudaliswaselevated

byCSD(Zhangetal.,2011).Theseinvivostudiesdidnotpresent anydataontrigeminalsensitizationbeyond120minafterCSD induction.ThepresentresultsindicatethatCSD-inducedtrigem- inalsensitizationwearsoffby48h.Importantly,wedemonstrated thatavoidanceofheadache-worseningfactors(lightandphysical

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activity)extendsbeyondtheperiodoftrigeminalsensitizationafter CSD.ThiscouldindicatethattheCSD-affectedmiceinourstudy wereconditionedtoassociatelightexposureandphysicalactivity withpain,ratherthantoavoidmovingbecauseofactualpain.The saliencenetworkplaysakeyroleintheevaluationofthestateor conditionofathreattoanorganism(Borsooketal.,2013).Ithas beenreportedthatthefunctionofthesaliencenetworkisaltered in actualmigrainesufferers(Malekietal.,2012)andaninﬂam- matorysoup-basedratmigrainemodel(Becerraetal.,2017).Our ﬁndingscouldthereforebeexplainedbydysfunctionofthesalience network.

Thetimepointsselectedforthepresentstudy(48hand72h afterCSDinduction)maycapturetheconditionthatisanalogous tothepostdromalphaseofmigraine.Photophobia,tiredness,and weaknessarefrequentlyobservedinthepostdromeofmigraine (Blau, 1982; Giffin et al., 2016; Kelman, 2006; Ng-Mak et al., 2011;Quintelaetal.,2006);tirednessandweaknesscanreduce physical activity,and most patientsfind postdrome symptoms debilitating(Blau,1982;Giffinetal.,2016;Kelman,2006;Ng-Mak etal.,2011;Quintelaetal.,2006).Photophobiaandhypoactivity impair qualityoflife andleadtoreduced productivityatwork.

In particular,photophobia is likely to interfere withworkthat requirestheuseofacomputerscreen(Chuetal.,2011;McAdams etal.,2020;Mullenersetal.,2001;Perenboometal.,2018).Cur- rently, thereisnoestablishedtherapyforthesesymptoms.Our datademonstratedthatpretreatmentwithNeurotropin^® at0.27 NU/kg reversedCSD-induced decreasesinambulatory timeand distanceinthelightzone.Adoseof0.27NU/kgisusedfortreat- ing neuropathic pain in humans (Hata et al., 1988). Thus, the optimal therapeuticdoseof Neurotropin^® for neuropathic pain maybeeffectiveformigraine-associatedphotophobiaandhypoac- tivity postdrome. Our ﬁndings can beclinically translated into situations in which Neurotropin^® pretreatment can ameliorate hypoactivity in a light environment in the postdrome period, thus allowing patientstoregain functionality and productivity.

It remains unknownhow Neurotropin^® improved photophobia and hypoactivityin ourmigraine model. The biological actions ofNeurotropin^®includesuppressionofproinﬂammatorycytokine production(Fukudaetal.,2020;Nishimotoetal.,2016),enhanced serotoninand noradrenergicactivity(Okazakiet al.,2008),glycosaminoglycan synthesis (Sakaiet al., 2018), and inhibition of nuclear factor-kappa B (NF-␬B) and mitogen-activated protein kinasesignalingpathways(Zhengetal.,2018).Intriguingly,arecent studyreportedtheinvolvementofNF-␬BandNF-E2-relatedfactor 2pathway-mediatedneuroinﬂammationinnitroglycerin-induced migraine-likesymptomsandphotophobia(Casilietal.,2020).As mentionedabove,migrainesufferersoftencomplainofphotopho- biaandreducedphysicalactivitythroughouttheinterictalperiod (Bondetal.,2015;Chuetal.,2011;McAdamsetal.,2020;Mulleners etal.,2001;Perenboometal.,2018;Rogersetal.,2020).Itwouldbe worthexploringwhetherchronicadministrationofNeurotropin^® improvesthesedisablingconditions.

Thepresentstudyhasseverallimitationsthatshouldbenoted.

First,theCSD-basedmigrainemodelmimicsmigrainewithaura attacks, andsoitremainsunclearwhetherourﬁndingsarealso applicabletomigrainewithoutaura.Second,theinvasivenature of thediseasemodelmeansthatcomparisonsrelevanttophys- ical activity wereonly made between CSD-subjected miceand sham-operatedmiceatidenticaltimepointsinthepresentstudy.

Asdescribedinthematerialsandmethodssection,electrodeinser- tionintothebraintissuewasnotdoneintheshamsurgery,because we observedthatelectrodeinsertioncouldinduceCSDinciden- tally.Third,migraineisapproximatelythreetimesmoreprevalent in women that in men (Dodick, 2018a). Nevertheless, we only usedmalemicetoexcludethepotentialeffectsofmenstruation onbehavior(Ebineetal.,2016).Arecentpaperreportedthatthere

isnosigniﬁcantsexdifferenceinCSD-inducedbehavioralpatterns (Harriottetal.,2021).Finally,asstatedabove,wedidnoteluci- datethemechanismbywhichNeurotropin^®amelioratedphysical activityinCSD-affectedmice,whichshouldbeclariﬁedinfuture studies.

Insummary,ourexperimentalmodelprovidesaplatformfrom whichtofurtherinvestigatepostdromalphotophobiaandhypoac- tivityinmigraine.Ourﬁndingsofferanoveldirectionforfuture migraineresearch.Furthermore,Neurotropin^®mayconferprotec- tionagainsttheseunrecognizedand undertreated symptomsat adoseusedfor themanagement ofneuropathicpaininclinical settings.

Authorcontributions

Mamoru Shibata: Conceptualization, Methodology, Format analysis,Investigation,Writing-OriginalDraft,Supervision,Fund acquisition.

SatoshiKitagawa:Investigation,Writing-Review&Editing.

ChunghuaTang:Methodology,Formatanalysis,Investigation, Writing-Review&Editing,FundAcquisition.

MiyukiUnekawa:Investigation,Writing-Review&Editing.

YoheiKayama:Investigation,Writing-Review&Editing.

JinNakahara:Writing-Review&Editing.

NorihiroSuzuki:Writing-Review&Editing,FundAcquisition.

Funding

This study was supported by JSPS KAKENHI [grant number 19K07849,toMS];agrant fromtheTakedaScienceFoundation toMS;and researchgrantsfrom PﬁzerInc.[WS1878886], Nip- pon Zoki Pharmaceutical Co., Ltd, and Kao Corporation to our researchgroup.ThisworkwassupportedinpartbyaJapan-China SasakawaMedicalFellowship[grantnumber2017816]andaState ScholarshipFundoftheChinaScholarshipCouncil[grantnumber 201908500072]toCT.

DeclarationofCompetingInterest

Theauthorsdeclarethattheyhavenoconﬂictofinterest.

Acknowledgements

WewouldliketothankDr.TsubasaTakizawaatDepartmentof Neurology,KeioUniversitySchoolofMedicine,andMr.Yasunao IshidaatNipponZokiPharmaceuticalCo.,Ltd.fortheiradviceand insightfuldiscussions.

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