Dunham, James P. and Hulse, Richard P. and Donaldson, Lucy F. (2015) A novel method for delivering ramped cooling reveals rat behaviours at innocuous and noxious temperatures: a comparative study of human psychophysics and rat behaviour. Journal of Neuroscience Methods, 249 . pp. 29-40. ISSN 1872-678X

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Anovelmethodfordeliveringrampedcoolingrevealsratbehavioursatinnocuousand

noxioustemperatures:Acomparativestudyofhumanpsychophysicsandrat

behaviour.

JamesP.Dunham1,RichardP.Hulse

1,2andLucyF.Donaldson

1,3.

1.SchoolofPhysiologyandPharmacology,MedicalSciencesBuilding,UniversityWalk,Universityof

Bristol.

2.CancerBiology,SchoolofMedicine,UniversityofNottingham,Nottingham.NG72UH(present

address).

3.SchoolofLifeSciencesandArthritisResearchUKPainCentre,UniversityofNottingham,University

Park,Nottingham.NG72RD(presentaddress).

CorrespondingAuthor:

JamesP.Dunham.HonoraryResearchAssociate,SchoolofPhysiologyandPharmacology,Medical

SciencesBuilding,UniversityWalk,UniversityofBristol.

Jimdunham@doctors.org.uk

Numberofpages33

NumberofFigures:6

NumberofTables:0

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Abstract

Background

Thermalsensorytestinginrodentsinformshumanpainresearch.Thereareimportantdifferencesin

themethodologyfordeliveringthermalstimulitohumansandrodents.Thisisparticularlytrueincold

painresearch.Thesedifferencesconfoundextrapolationandde-valuenociceptivetestsinrodents.

NewMethod

Weinvestigatedcooling-inducedbehavioursinratsandpsychophysicalthresholdsinhumansusing

rampedcoolingstimulationprotocols.APeltierdevicemounteduponforcetransducers

simultaneouslyappliedarampedcoolingstimuluswhilstmeasuringcontactwithrathindpawor

humanfingerpad.Ratwithdrawalsandhumandetection,discomfortandpainthresholdswere

measured.

Results

Rampedcoolingofarathindpawrevealedtwodistinctresponses:Briefpawremovalfollowedbypaw

replacement,usuallywithmoreweightbornethanpriortotheremoval(temperatureinter-quartile

range:19.1oCto2.8

oC).Fullwithdrawalwasevokedatcoldertemperatures(interquartilerange:-

11.3oCto-11.8

oC).Theprofileofhumancooldetectionthresholdandcoldpainthresholdwere

remarkablysimilartothatoftheratwithdrawalsbehaviours.

Comparison

Previousratcoldevokedbehavioursutilisestatictemperaturestimuli.Byutilisingrampedcoldstimuli

thisnovelmethodologybetterreflectsthermaltestinginpatients.

Conclusion

Briefpawremovalintheratisdrivenbynon-nociceptiveafferents,asistheperceptionofcoolingin

humans.Thisisincontrasttothenociceptor-drivenwithdrawalfromcoldertemperatures.These

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findingshaveimportantimplicationsfortheinterpretationofdatageneratedinoldercoldpainmodels

andconsequentlyourunderstandingofcoldperceptionandpain.

Keywords:pain,behaviour,rat,human,cold,nociception

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1 Introduction

Coolingevokedbehavioursinrodentsareoftenstudiedwiththeaimofinformingourincomplete

understandingofthermalperceptioninhumans.Thiseffortishamperedbysignificantdifferencesin

themethodologyusedwiththedifferentspecies.Theutilityofresultsgeneratedinrodentswith

respecttotheaboveaimcan,therefore,bequestioned.Thusthereisaneedtobetterunderstandthe

relationshipsbetweencoolingevokedbehavioursinrodentsandcoolingevokedsensationsinhumans.

Inhumanpsychophysicalexperiments,rampedcontactcoolingisdeliveredviaathermode,usually

usingdevicessuchasthosemadebyMEDOC(http://www.medoc-web.com/medoc_en_home.aspx)or

SOMEDIC(http://en.somedic.com/default.asp?pid=29).Importantly,applicationoframpedcoolingto

skinenablesdeterminationofthresholdsforcolddetectionandpaininhealthyindividualsand

subsequentlythedefinitionofpositiveandnegativesymptoms/sensationsinpatients[1,2].

Contactthermalstimuli,oftenwithvaryingrampratesandfinaltargettemperatures,arealsousedin

pre-clinicalelectrophysiologicalexperimentsinsensoryprimaryafferentresearch[3-6].Thisenables

captureofthermalthresholdstoneuronalactivationandtheresponsestosuprathresholdstimuli.

Incontrast,behaviouralexperimentationinanimalsutilisessignificantlydifferentcoldstimuli.These

include;coldplates(statictemperature),evaporativecooling,orplacepreferencetests[7-10].The

mostcommonoutputsaretime-related,e.g.latencytobehaviour(pawwithdrawal[11]lickingor

flinching),behavioursperunittime[7,12]ortimespentinaparticularlocation.Itisrarethatthermal

behaviouralthresholdspersearemeasuredinanimals,althoughdecreasingtemperatureplateshave

beendescribed[13],enablingdifferentiationbetweencoldallodyniaandcoldhyperalgesia.Most

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thermaltestsmeasurehyperalgesia,butmilderinterventionssuchasevaporativecoolingofacetone,

havealsobeenusedtoelicitallodynicbehaviours[8,14].

Afurtherimportantconsiderationisthatasrampedcoolingisalmostneverusedinrodentstudies

therearenodataregardingbehaviourselicitedduringthetransitionbetweeninnocuousandnoxious

cooling.Giventhatanyputativebehavioursarelikelytobedifferentandthatthistransitionmustoccur

(albeitmorerapidly)whenusingroutinecoldstimulisuchascoldplates;moredetailedconsideration

ofsuchbehavioursandtheimpactthattheymayhavehadontheinterpretationofpreviousworkis

warranted.

Furtherstill,mostcoolingevokedbehavioursareelicitedviacoolingdeliveredtoallfourpaws;thetail

andalsotheabdomen(andtestesinmales).Thus,inadditiontonociceptivereflexesthatmaybe

present,theobservedbehaviourswillalsobeinfluencedbymechanismsinvolvedinmaintainingbody

temperature(homeostasis)[15]andmaybecomplicatedbyfear/avoidancebehavioursevokedbyan

inescapable,potentiallynoxiousstimuli.TheHargreavestest[11,16],byrestrictingheatstimulationto

asinglepaw,removedtheseconfoundsinheatnociceptivetesting.

Theaimofthisstudywastodevelopmethodologytoaddressthesediscrepanciesbetweenrodentand

humancoldtestinginordertobetterapplyknowledgegainedinthelaboratorysettingtohumancold

perceptionandpain.Toachievethis,thesamecoolingstimuluswasusedtoevokebehavioursin

healthyratsandsensationsinhealthyhumans.Wedeliveredrampedcoolingtoasinglerathindpaw

anddeterminedcontacttemperaturesatwhichcoolinginducedbehavioursoccur.Thismethodwas

usedinparalleltodeterminecooldetection,colddiscomfortandcoldpainthresholdsinhealthy

humans.

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2 MaterialsandMethods

2.1 Apparatusfordeliveringrampedthermalstimulitoglabrousskininhumansandrats

Theapparatususedinallexperimentswasdesignedandbuiltin-house,astherewasnoavailable

commercialapparatusthatcouldreproduciblyandreliablystimulatebothratandhumanglabrousskin

toenablemeasurementofequivalentthermalthresholds.

APeltierunit(Supercool,Gothenburg,Sweden,(cat.no.131-10-13);40mmx23mmx3.6mm)was

attachedwiththermallyconductiveadhesive(ArcticSilver5,fromArcticSilverCA.USA)toan

aluminiumheatsink.ThePeltierandheatsinkmodulewasmounteduponforcetransducers(FSseries

ForceTransducer,RadiosparesUK,catno.235-6210),whichenabledmeasurementoftheprecise

temperatureatwhichtheforcetransducerswereunloaded.Thesurfacetemperatureoftheunitwas

recordedviaaTTypethermocouplemountedonacopperplateaffixedwithArcticSilverTMtothe

uppersurfaceofthePeltierdevice.Duringcooling,theheatsinkwasflushedwith50:50ethylene

glycol:water,pre-cooledto-10oC.Thisenabledrapidandlargereductionsintemperature.During

heatingexperiments,theheatsinkwasflushedwithcoldwater.ThePeltierdevicewasdrivenfroma

controlsystembuiltinhouse,whichenabledfinecontroloflinearheatingandcoolingrates.

Thermocoupleandforcetransduceroutputswereamplifiedandfedintoamicro1401analogueto

digitalconverter(CambridgeElectronicDesign).DatawererecordedonaPCusingSpike2v6

(CambridgeElectronicDesign)forsubsequentofflineanalysis(Figure1).

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2.2 Determinationofthermalthresholdsinrats

AllexperimentsinvolvedmaleWistarrats(250-350g,Harlan,UK)andwerecarriedoutwithUniversity

ofBristolEthicalReviewPanelapprovalandinaccordancewiththeUKAnimals(ScientificProcedures)

Act1986.ThismanuscriptwaspreparedwithreferencetotheARRIVEguidelines[17].Animalswere

housedinenrichedenvironments,under12:12hourlight:darkconditionsandhadadlibitumaccessto

foodandwater.Priortoexperimentaltesting,animalswerehabituatedtoboththetestingapparatus

andtotheinvestigators.

RatswereplacedinaPerspexenclosuresimilartothatusedinLintonInstrumentation’sIncapacitance

tester(http://www.lintoninst.co.uk/).Theysettledintoapositionsuchthatthehindpawswerein

contactwiththePeltierdevice(Figure1A,C).Occasionallyitwasnecessarytomakeminoradjustments

tothepositionofthepaws.Oncetheanimalwassettled,thermalstimuliwereappliedtotheglabrous

hindpawskin,fromaholdingtemperatureof25oC.Inthreerats,heatingwasdeliveredatarateof

1oC/s,untilwithdrawal,uponwhichtheplatetemperaturewasreturnedtobaseline.Thisprocesswas

repeatedupto4timesperrat.Intenrats,coolingwasdeliveredatarateof-1.3oC/sandcontinued

untiltheanimalshiftedtheweightoffthecooledhindpaw,denotingwithdrawalfromthestimulus.

Thelowestachievabletemperaturewas-12oCwhichwasthereforeeffectivelythecut-offtemperature.

AfterstimulationthePeltierdevicetemperaturewasreturnedtobaseline.Asecond,andoccasionallya

third,rampwasthendeliveredwithaninter-stimulusintervalnolessthan3minutes.Occasionallyit

wasnecessarytorepeatrampsif,forexample,theratturnedaroundintheboxthusremovingthe

hindpawfromcontactsurface.

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Fouradditionalratsweretestedwithvariablecoolingratesof-0.5,-1,-2and-4oC/s.Otherthanthe

differentrates,theseexperimentswereperformedasdescribedabove.

Attheendofthebehaviouraltestingthepawsoftheratswereinspectedforsignsofinjuryincluding

erythemaandoedema.

2.3 Relationshipbetweensurfaceandsubcutaneoustemperatures

Behaviouralwithdrawaltocontactheatingisknowntodependonthesubcutaneousheatingrate[18].

Itwasthereforenecessarytoevaluateboththerateofsubcutaneouscoolingandtheabsolute

subcutaneoustemperatureachievedduringtherodentexperiments.Inordertodeterminethe

subcutaneoustemperaturesatwhichcoolingbehavioursoccurred,oneanaesthetisedadditionalrat

wasused.Anaesthesiawasinducedandmaintainedwithsodiumpentobarbitone(induction:60mg/kg

intraperitoneal,maintenance:20mg/kg/hr(intravenous)andthetracheawascannulatedforairway

maintenance.AT-Typethermocouple(madein-house)wasinsertedsubcutaneouslyintotheplantar

skinofonehindpaw.Theanaesthetisedratwasthenpositionedintherestrainingbox(Fig1C)with

hindpawsfirmlyincontactwiththePeltierunit,andthePeltierdevicewascooledatdifferentrates.

Thesubcutaneouspawtemperaturesthatcorrespondedtothecontacttemperaturesweredetermined

foreachramprate.

2.4 Determinationofthermalthresholdsinhumanvolunteers

ThestudywasgivenethicalapprovalbytheFacultyofMedicalandVeterinarySciencesCommitteefor

ResearchEthics,UniversityofBristol.Participantsgaveinformedconsentpriortotesting.

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Participantswereexcludedfromthestudyiftheysufferedanyneurologicalorotherproblemsthat

couldaffecttheirabilitytodetectorrespondtocutaneousthermalnoxiousstimuli.

Tenhealthyparticipants(5male,5female,26.4yrs±1.2(meanage±SEM))wererecruited.No

participantwassubsequentlyexcludedforanyreason.

Participantswereacclimatisedtothetestingfacilitypriortotesting.Theprotocolwasexplainedto

themsotheywereawareoftheprocedure,andtheywereexposedtoafamiliarisationramppriorto

testing.

ParticipantswereaskedtoplacethepadoftherightindexfingeronthePeltiersurface,whichwas

initiallyheldat30oC.Theinstructionsweretorest/placethefingertipuponthecoolingsurfaceand

nottoexertanyspecificforce[19].Aftermorethan10secondshadelapsed,theinvestigatorinformed

themthattherampwouldbeginwithinthefollowing10seconds.Participantswereaskedtosaywhen

theydetectedthetemperaturechange(detection)andwhentheydetectedthetransitionintoan

uncomfortablesensation(discomfort).Inputfromafootpedalwasusedtocapturedetectionand

discomfortthresholds.Participantswereinstructedtoremovetheirfingerfromtheequipmentwhen

thesensationbecamepainful;thiseventwasrecordedviatheforcetransducer.Followingwithdrawal

theequipmenttemperaturewasreturnedto30oC.Theheatingratewas~1

oC/s,whereascoolingrate

was-1.3oC/s.Thesamerampwasappliedthreetimes.

Immediatelyaftereachtest,participantswereaskedtochoosetwodescriptorsfromapredefinedlist

thatindicatedthebestdescriptionofthesensation(s)theyexperiencedatwithdrawal(i.e.thequality

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ofthepain).Thedescriptorslistwasconstructedwithreferencetopreviouspsychophysicalstudies

[20-22].Fourparticipantsfounditdifficulttogivetwodescriptorsforeachramp.Whenthisoccurred,

thesingledescriptorornodescriptorswererecorded.Oneparticipantforgottoprovidedetection

thresholdson2of3ramps.Tokeeptestingnumberconsistentbetweensubjects,andtocomplywith

ethicscommitteeapproval,theserampswerenotrepeatedinthissingleindividualanddatawere

includedasanincompleteset.

2.5 Statisticalanalysis

Prism4forWindows(Version4,Graphpad)andSPSS(version18;IBM,Armonk,NY)wereusedfor

statisticalcomparisons.Dataarepresentedorshownasmean±standarderrorofthemean(SEM)or

median(interquartilerange(IQR))asstated.Thresholdandwithdrawallatencyatinitialandfull

withdrawal(rats)werecomparedusingMannWhitneytests.Meantemperaturesandlatenciesfor

evokedbehavioursatdifferentcoolingrates(rats)andhumanpsychophysicalandratbehaviouraldata

werecomparedusinganon-parametric1wayANOVA(KruskalWallis)followedbyDunnsposthoctest.

Thresholdsinhumanparticipants(colddetection,discomfortandnoxiouswithdrawal)werecompared

using1wayrepeatedmeasureANOVAfollowedbyBonferroni’sposthoctest,unlessexplicitlystated.

Wheninvestigatingtheeffectofcoolingrateonevokedbehaviours,alineartestfortrendwas

additionallyperformedasaposthoctestfollowing1wayANOVAanalysisofthelog-logtransformsof

thelatenciestoinitialandfullwithdrawalatthedifferentrates.

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Subcutaneoustemperaturesduringcoolingatwhichinitialandfullwithdrawaloccurredwere

interpolatedofflinefromthedatameasuredinun-anaesthetisedrats.

Hierarchicalclusteringofthelatenciesofallbehavioursinducedduringcoolingat-1.3oC/swas

performedusingtheWardmethodandtheSquaredEuclideandistancemeasureinSPSS.Theoptimum

numberofclusterswasdeterminedviaexaminationoftheagglomerationcoefficients.SPSSwasthen

usedtoassignclustermembershiptotheindividuallatencies.Frequencyhistogramsofcluster

membershipswerethengeneratedusingPrism.UsingSPSS,atwo-clustersolutionwasthenappliedto

temperaturesofallbehavioursinducedbycoolingat-1.3oC/s.Thefrequencyhistogramsofbehaviours

vstemperaturewerethengeneratedinPrism.Itshouldbenotedthatclusteranalysisdoesnotprovide

astatisticthatcanbeusedtodeterminetheprobabilityofthedatasetcontainingacertainnumberof

clusters.

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3 Results

3.1 Heatingresponsesinratsandparticipants.

Heatingofthefingerpadoftheindexfingerofthehumanparticipantseliciteddetection,discomfort

andwithdrawalthresholdsof38.1oC±1.5

oC,47.4

oC±1.2

oCand49.8

oC±0.9

oC(Mean±SEM)

respectively(Figure2A,2C).Heatingoftheplantarsurfaceofasinglerathindpawelicitedarobust

withdrawalresponseat47.8oC±0.4

oC(Mean±SEM)(Figure2B,2C).Whilsthumandetection

thresholdsoccurredatsignificantlylowertemperaturesthanratwithdrawal,nodifferenceswere

foundbetweenhumandiscomfort,humanwithdrawalandratwithdrawaltemperatures(Figure2C).

3.2 Coolingresponsesinrats

Coolingstimuliwereappliedfollowingtheabovevalidationofthismethodofdeliveringanisolated

thermalstimulustoasinglerathindpaw.Coolingofonehindpawelicitedtwodistinctbehavioursin

therat.Thesebehaviourswereidentifiedbyobservationandcouldalsobeseenintheweightbearing

profile,shownasrawdatainFigure3A.Initialbriefpawremovals,thatwerenotsustained,were

usually,butnotalways,seenasthecontacttemperaturedecreased.Thisinvolvedabriefremovalof

thepawfromtheplate(1-2sduration),whichwasthenreturned,oftensubsequentlybearingmore

weightthanbeforedespitethecontinuedloweringofcontacttemperature(Fig.3A).Thisincreasein

weightbearingonthestimulatedpawwasnotseenintheresponsestoheating.Asthetemperature

continuedtodecrease,afullwithdrawalwasevokedasthepawwasremovedfromtheplateand

remainedlifteduntiltheplatewasre-warmed(Fig.3A).Whenpawremovalsweregroupedinto

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”initial”iftheywerenotsustained,and”full”whenthepawliftwassustainedasdeterminedbythe

trace,theninitialbriefandfullremovalsoccurredatcontacttemperaturesof14.3oC(2.8

oCto19.1

oC)

and-11.7oC(-11.3

oCto-11.8

oC)respectively(median(inter-quartilerange)).Fullwithdrawaloften

occurredattemperaturesapproachingthelowestthatwereachievable(approximately-12oC,Fig.3B).

Thedifferencesinwithdrawaltemperatureswerereflectedinwithdrawallatencies(initial11.6s(8.2s-

20.0s),fullwithdrawal33.5s(31.3s-36.4s)).Thevariabilityinthelatencytofullwithdrawalwasgreater

thanthatseeninthevariabilityintemperatureatfullwithdrawal.Thisprobablyreflectsevents

occurringaftertheattainmentofthelowestpossibleplatetemperature.Thelatencieswerealso

significantlydifferentbetweeninitialandfullwithdrawalvalues(Fig.3C).

Itwasevident(Figs.3B,C)thatthetemperaturesatwhichinitialpawremovaloccurredoverlapped

withthosetemperatureselicitingfullwithdrawal.Todeterminewhetherhindpawcoolingdidindeed

evoketwodistinctbehavioursintherat,whethertheinitialandfullwithdrawalswereacontinuumof

thesamebehaviour,orwhethersubjectiveexperimenteropinionwasinfluencinginterpretation,

latenciesandtemperaturesweresubjecttoclusteranalysis.Examinationoftheagglomeration

coefficientsforlatenciesmoststronglysupportedamodelwith2clusters,althougha3or4cluster

modelcouldnotbediscountedfromtheagglomerationcoefficientsalone.Examinationofdifferent

clustersolutions(i.e.2,3or4clusters)alwaysgeneratedafirstclusterwithamedian(I.Q.R)latencyof

11.7(8.6to14.3)seconds(initialremoval)andthisclusteralwaysincludedthesamenumberofdata

points,suggestingthatthetwobehaviourswereindeeddistinct.Usingatwoclustermodelgavea

median(I.Q.R)latencyforthesecondclusterof28(23.4to31.7)seconds.Threeandfourcluster

modelsallsplitthelongerlatencycluster(fullwithdrawal)intoadditionalsubclusterswithnoeffecton

thefirstcluster(Fig.3E).Furthermore,thefirstclusterinthetwoclustersolutionfortemperaturevs.

responsescontainedasimilarnumberofresponsesasthefirstclustergeneratedusinglatencies:38

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datapointsfortemperaturesvs.36datapointsforlatencies.Thisgeneratedafirstclusterwitha

median(I.Q.R.)temperatureof13.6(18.8to10.3)oCandasecondclusterwithamedian(I.Q.R.)

temperatureof-8.1(-2.5to-12.3)oC(Fig.3D).

Therateofcontactheatingandsubsequentsubcutaneousheatingrateinratsaffectsdifferentgroups

ofnociceptiveafferents[18].Wethereforedeterminedtheeffectofdifferentcoolingratesonthe

evokedratbehaviours.

Increasingtherateofcoolingfrom-0.5to-4oC/sreducedthelatencytobothinitialbriefremovaland

fullwithdrawal(Figs4A&4B),butdidnotaffecttheskintemperatureatwhichinitialorfullremoval

occurred(Figs4C&4D).Interpolationofthesubcutaneoustemperatureatwhichbehaviourswouldbe

expectedtooccur,asderivedfromtheskinandsubcutaneoustemperaturesintheanaesthetisedrat,

generatedsubcutaneousmeaninitialremovaltemperaturesofapproximately5oCto10

oC(Fig.4E).

Thesewerehighlyvariable,asseenfortheskincontacttemperatures(Fig.3B).Notably,interpolated

subcutaneoustemperaturesatfullwithdrawalweremuchlessvariable,meanwithdrawal

temperatureswere~-2oC(Fig.4F).

Thesubcutaneoustemperaturewaslinearlyrelatedtothesurfacetemperatureandthislinear

relationshipwasidenticalforrateofcoolingfrom-0.5oC/sto-2

oC/s.Atthefastestrateofcooling,-4

oC/s,thiswasnolongerthecase,andsubcutaneoustemperatureswereslightlyhigherthanwouldbe

predictedfromalinearrelationship(Fig.4G).

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3.3 Coolingresponsesinparticipants

Inhumanstudies,participantsindicateddetectionanddiscomforttocoolingandthiswascapturedon

therawtraceviaafootpedalinput.Fingerremovaloccurredwhenthesensationbecamepainful(Fig.

5A).Coolingdetectionoccurredat24.9oC(26.3

oCto19.5

oC)(median(IQR)),discomfortat-1.2

oC(1.2

oC

to-5.9oC)andnoxiouswithdrawal-6.3

oC(-3.5

oCto-10.3

oC)(Fig.5B).Notably,coolingdiscomfortand

noxiouswithdrawalwerenotevidentuntilskincontacttemperatureswerelessthan0oC.Themost

frequentwordschosentodescribethesensationevokedatnoxiouscoldwithdrawalwere“cold”‚

“numbing”and“freezing”(Fig.4C).

Duetothemismatchbetweenhumanandrodentstudiesoncoldnociception,oneaimwasto

determinewhetherratbehaviourscouldberelatedtopsychophysicalcorrelatesinhumans.Asthere

werepotentialdifferencesinskinthicknessandthermaltransferbetweenratsandhumansandwe

couldnotdirectlymeasuresubcutaneoustemperaturesinhumans,andmanyratwithdrawalsoccurred

atthecut-offtemperaturewhereashumansdidnot,wecomparedlatencytowithdrawalratherthan

contacttemperatures.Comparisonshowedthatratfullwithdrawalandpain-evokedwithdrawalin

humansoccurredatasimilarlatencyinbothspecies(Fig.6A-C),indicatingthatthismethodof

stimulationelicitednociceptioninbothspeciesatequivalenttimesafteronsetofcoldramping.In

addition,initialremovallatenciesinratswereequivalenttocolddetectionlatenciesinhumans(Fig.

5C).Probablymostcrucialisthattheprofileofresponsestoreducingtemperatureiscomparable

betweenthetwospecies.

Interestingly,latencytonoxiouscoldwithdrawalinhumanswasnotclearlydistinguishablefrom

latencytocolddiscomfortinhumansorfromlatencytofullcoldwithdrawalsinrats.Therewasalsono

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statisticaldifferencebetweenlatenciestocolddetection/initialremovalsinhumansandrats

respectively(Fig.5C).Thissupportstheinterpretationthattheinitialpawremovalresponsesinratsare

associatedwithnon-noxiousratherthannoxiouscold.Thevarianceofbothcoldmeasures(initialand

fullwithdrawals)intheratwassignificantlygreaterthaninthehumanmeasures,i.e.initialrat

responsestorampedcontactcoldstimulationwerelessconsistentbetweentrialsthanthoseof

humans.

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4 Discussion

Theneurophysiologyofthermalperception,particularlythatofcoldpain,iscomplex.Multipleafferent

populationsandascendingpathwaysinformperceptionandbehaviouralresponses[23],and

processing,particularlyofnoxiouscoldinformation,isalsoinfluencedbydescendingmodulationfrom

brainstemcentres[24-26].Furthercomplexityisaddedtointerpretationoffindingsbymethodological

differencesbetweenstudies,includingstimuli(magnitude;area;skintype)andoutcomes

(psychophysics,behaviour,neuronalproperties,imaging).Overarchingalloftheabovearepossible

differencesbetweenspecies.

Thiscomplexsituationhasledtoassertionsandpossibleover-simplifications,which,especiallywhen

takenoutofcontextoftheoriginalwork,canbecontradictorytoeverydayexperience.Forexample,it

iscommonlyreportedthathumanshaveacoldpainthresholdofnearto14oC,afigurewhichhasbeen

usedtorelateperceptiontothefunctionofindividualputativetransductionmolecules[27].However,

itisnotusuallypainfultohandleabottleofmilkfromafridgeat4oCanditispossibletohandlefrozen

foodsfromadomesticfreezer(-20oC)forshortperiodsoftimewithoutsufferingpain,althoughitcan

beuncomfortable.

Wesoughttoaddressthemismatchbetweenexperimentalconclusionsinratsandhumans,and

everydayobservationsoncolddiscomfort/pain,andtocomparecoldbehaviouralresponsesinhumans

andratsbyusingequivalentcoldrampingstimuliinbothspecies.

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Initialexperimentssoughttovalidatethisnovelmethodofdeliveryofthermalstimuli.Thiswas

possiblewithreferencetopreviouslypublishedheatwithdrawalthresholdsinrats.Heatwithdrawal

thresholdsreportedhereofcirca48oCareremarkablysimilartothe47.6

oC±0.2

oCgeneratedutilising

thermalstimulifromaradiantheatinglamp[16],acomparablethermalstimulus.Furthermore,andas

notedbyBanikandKabadi,2013[16],thesewithdrawaltemperaturesarecomparabletothose

generatedinhumans(datahereinande.g.47.5oC,[28]).Theratheatthresholdsreportedhereare

somewhatlowerthanthosereportedusingasimilarmethodofmeasuringheathyperalgesiadescribed

byTaboandcolleagues(1998).ThismethodcombinedforcemeasurementwithaPeltierthermodebut

theforcetransducersrecordedtheforceofwithdrawalandnottheweightbornebythepaw[29].

Giventhesepositivevalidationexperiments,wewerethenabletoexploretheunknowneffectsofthe

applicationoframpedcoolingstimuli.Inequivalentskintypes,bothspeciesdemonstratedthatthey

wereabletorespondtotheonsetofcoolingfromambientatequivalentlatencies/temperatures,and

thisoccurredattemperaturesnotperceivedaspainfulbyhumans.Wethereforehypothesisethatthe

initialbriefpawremovalseeninratsisnotanocifensivebehaviour.Thishypothesisisstrongly

supportedbythenewobservation,whichisonlypossibletomakeusingthisnovelapparatus,that

moreweightisbornebythepawafteritisreplaced,duringtheseinitialbehaviours.

Itisinterestingtonotethatcoldinducedvasoconstrictionduringcoolingofisolatedpawsoccursinthe

ratatapproximately22oC[30].Itcouldbethatthebriefremovalfromthecoolingsurfacerepresentsa

homeostaticresponsetocooling,asrodentpawsandtailarecriticalformaintenanceofbody

temperatureintherat[31].Intherat,withoutthebenefitoftheadditionalinformationonincreased

weightborneonthecooledhindpawafterthesemovements,themoretransientbehavioursevoked

bynon-noxiouscoolingcouldeasilybemisinterpretedasafullwithdrawalresponse.Itonlybecomes

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apparentthatwithdrawalsaretransientwhenappliedtemperaturesaresubsequentlyloweredto

levelswherecompletewithdrawaloccurs.Ifatrialisterminatedoninitialpawremoval,thecold

thresholdwillberecordedatahighervalue.Indeed,noxiouscoldthresholdsinratsareoftenquotedat

valuesmuchwarmerthanthosewereport(e.g.circa4or5oC[7,12]),intherangeinwhichmostofthe

briefpawremovalsoccurred.

Theinitialpawremovalisclearlydifferentfromtheovertfullwithdrawalsevokedbylowercontact

temperaturesthatmostobviouslycorrelatewiththenoxiouscoldthresholdsmeasuredinhumans.

Suchwithdrawalsoccurattemperaturessimilartothosethatevokewithdrawalinlightlyanaesthetised

animalswhich,bydefinition[32],arenociceptordriven[24,33].Thisfullwithdrawalbehaviourand

thecorrelateinhumansisthereforemorelikelytobedrivenbycoldnociceptors,andmaybean

appropriatemeasuretouseinpreclinicalcoldevokedpainresearch.Itshouldalsobenotedthatfor

bothbriefinitialpawremovalandnoxiouswithdrawals,theresponsesinratsaremuchmorevariable

thanthoseelicitedinhumans.Itisthereforecleartousthattheinterpretationofcold-evoked

withdrawalbehaviourinrodentsiscomplicatedbythemorecomplexwithdrawalbehaviourthanis

seenwithheatstimulation.

Asrateofcoolingincreased,latencytobehavioursdecreased,yetthetemperaturesatwhich

behavioursoccurredwasconsistent.Thisfindingsuggeststhatitistheabsolutetemperatureatthe

neuronalreceptor(subcutaneous/dermal/epidermaletc.)thatdrivesbothinitialandfullwithdrawals,

ratherthantherateofchangeofcutaneoustemperature.

Subcutaneoustemperaturemeasurementsindicatethatfullnoxiouswithdrawalbehaviours,inratsat

least,areevokedattemperaturesjustbelow0oC,withcontact/surfacetemperaturewellbelow0

oC.It

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iswellknownthatfreezingispainfulandmayleadtotissuedamage[34].Whetherthewithdrawal

behaviourdescribedherereflectsactualfreezingpainisdebatable.Therewasnoevidenceoftissue

damagetothepawaftertestingandnosensitisationofresponseswasseen,eventhoughbothwould

beexpectedfollowingtissueinjury.Furthermore,itmaybearguedthatnociceptivesystemsfunction

towarnofthepotentialforinjuryandthatthissystemwouldacttopreventsuchinjuryinanawake

animal.Itisalsoknownthatthermoregulatorymechanismsarebluntedunderanaesthesia[35]andit

isthereforepossiblethatthesubcutaneoustemperatureinawakeanimalswerenotaslowasinthe

anaesthetisedrat.

Whileitisdifficulttocompareourdatatopreviousworkbecauseofthedifferencesinmethodology,it

isinterestingtonotethatpreviouslyreportedcoldpainthresholdsinbothratsandhumansoccurred

atwarmertemperaturestothosereportedhere.Onthethenareminence,thresholdsarereportedas

between10oCand15

oC[20-22,36,37]thoughwithhighvariability[38],aswithotherskinsites,

whereasourdatashowthresholdssubstantiallylessthanzerooCinbothspecies.Wesuggestthatthese

differencescouldbeaccountedforbyacombinationoffactors,includinglackof

expectation/anticipationeffects[39,40]whenusinganescapablestimuluswhereterminationis

entirelyundertheparticipant’scontrol;asmallerstimulusareaandthereforereducedspatial

summation[41],andthelowercoldsensitivityofglabrousskin[21],inparticularthefingertipin

relationtootherskinsites[38].

Theaimofthisstudywastodevelopmethodologytoaddressdiscrepanciesbetweenrodentand

humancoldtestinginordertobetterapplyknowledgegainedinthelaboratorysettingtohumancold

perceptionandpain.Therearehoweverknowndifferencesbetweencool/coldprimaryafferentsin

rodentsandprimates.Asmentionedabove,whiledifferencesinstimulationprotocolsand

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preparationscomplicatecomparisonsbetweencutaneousafferents,similarpatternsofresponsesdo

emerge.Moststudiesreporttwomajorcoldsensitiveafferentpopulationsintherat,unitswithtonic

activityattheholdingtemperatureandpolymodalnociceptiveunitsthatalsorespondtomechanical

andoccasionallyheatstimuli[42-44].

Tonicallyactive“cool”unitsinratshavemaximaldischargeratesatvariabletemperatures,depending

onstimulationprotocol.Stepandholdprotocols,withaholdingtemperatureof32oCandcoolingto

12oC,showmaximaldischargeratesbetween27

oCand17

oC[42].Incomparison,arampprotocol

similartothatreportedherein(holdingtemperature30oC,-1

oC/srampcoolingto0

oC),demonstrated

maximaldischargeratesbetween10oCand25

oC[44]).Thesetonicallyactiveunitsrepresentonefifth

toonequarterofthesampledpopulation,andhavepredominatelyCfibres,withonlyafewconducting

intheAδrange[42-44].Anoticeabledifferencebetweentheseratcoolingreceptorsandthosein

primatesisthatprimatetonicallyactivelowthresholdcoolreceptorsconductintheAδrange[23,45].

AlthoughmodellingandischaemicblockssuggestthattheseAδafferentsarelikelytoberesponsible

fortheperceptionofcoolinhumans[37,38,46],therearealsoanumberofC-fibreafferentsthat

behaveverymuchlikethosefoundintherat(maximumfiringataround15oC),inhumans[47].

Incontrasttocoolingafferents,nociceptiveafferentsaresilentatnormalskin/holdingtemperatures

(e.g.30oC),havebothCandAδfibres,andreportedcoldthresholdsof12

oCto22

oC[42,44].Mostof

thesestudiesdonot,howeverexaminetheresponsesoftheseprimaryafferentstosub-zerocoolingin

vivo.AllnociceptiveCandAδafferentswereexcitedwhencoolingtosub-zerotemperatures

(thresholdsbetween12oCand-6

oCandbetween0

oCand-12

oCrespectively)[5].AllAδnociceptors

respondedtolowtemperaturecoolingandencodedstimulusintensity,bothintermsofafferent

recruitmentandfiringfrequency,downtoatleast-12oC[6].Asignificantproportion(~40%)ofhuman

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C-fibrepolymodalnociceptorsalsorespondtocoldtemperatures(between19oCand0

oC).Itisalso

interestingtonotethatthissecondpopulationhadaveryvigorousresponsetofreezing[48,49].

Withthesedifferencesandsimilaritiesinratandhumancoldafferents,wespeculatethatcold

detectioninhumansismediatedbyAδfibresbutinrat,manifestedbytheinitialwithdrawals,itis

mediatedbythefunctionallysimilarratCfibrecoolfibres,basedonthetemperaturesatwhichthese

fibresshowmaximalfiring[23,37,38,45,46].Thediscomfortandpainfulwithdrawalinbothspecies

wouldappeartobemediatedbythenociceptiveAδfibreafferentsthatareactivatedatsubzero

temperatures,butthereisprobablyalsoacomponentfrompolymodalCfibrecold-responsive

nociceptors[5,6,48,49].

Toestimatethetemperaturechangeatthereceptorterminal,weinterpolatedthesubcutaneous

temperaturechangefromdataobtainedinanaesthetisedrats.Thetemperaturechangeattheafferent

receptorsisdependentnotonlyupontheappliedtemperatureofthePeltierdevice,butalsoother

factorssuchascontactpressure,bloodflowandotherphysicalandphysiologicalcompensatory

mechanisms[19].Contactpressureincreasedasratsshiftedmoreweightontothecooledpawafter

theinitialwithdrawals.ThiscouldincreasetheskinareaincontactwiththePeltier,possiblyreducing

thedistancebetweenreceptorandPeltierbycompressingtheskin.Localbloodflowcouldbereduced

bythisbehaviour,whichwouldalsothenaffectheat/coldtransfer,andsoincreasedpawpressure

couldincreasetissuecooling.Itispossiblethatthisincreasedcontactoninitialisinvolvedinimproving

coolingdetection.Interestingly,thepawpressureincreaseswerenotseenduringtheheating

experiments,indicatingacooling-specificbehaviour,ratherthannon-specificresponsetopaw

temperaturechange.Inhumans,weattemptedtomitigateasmanyofthesepotentialconfounding

factorsaspossible,byinstructingparticipantstoplacetheirfingeronthetestingdevicewithonlylight

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contactpressure.

Finally,thereareobviouslyinherentdifficultiesinrelatingpsychophysicaloutcomesinhumansand

behaviouraloutcomesinrats,notleastthatratslacksomeneuroanatomicalstructureswithinthe

forebrainthatarecrucialtothehumanexperienceofpain[50].However,withdrawalbehavioursinthe

rataredrivenbyafferentpopulationsthatappeartohaveequivalentcounterpartsinhumans[42].

Giventhatamajorreasontoundertakeresearchinexperimentalanimalsistoinformandimprove

humanpainmanagement,webelievethatthetwospeciesshouldbeinvestigatedinparallel,to

highlightsimilaritiesanddifferences.

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5 Conclusions

Wehaveusedanovelmethodofmonitoringbehaviouralandpsychophysicalresponsestocoolingand

noxiouscoldinbothratsandhumans.Bydeliveringrampedcoolingourtechniquemoreclosely

reflectscurrentlyusedclinicalprotocolsandthusfacilitateshumancomparativestudies.Thisapproach

hasrevealedacoolinginducedbehaviourintheratthatisunlikelytobedrivenbynociceptive

afferents.Ithasalsorevealedthatfullwithdrawalfromcoldstimulirequiresmuchcolder

temperatures,andthussubcutaneoustemperatures,thanhavepreviouslybeenemployed.Thiswork

hasimportantimplicationsfortheinterpretationofpastandongoingworkstudyingphysiologicaland

pathophysiologicalcoldpain.

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6 Acknowledgements

Wegratefullyacknowledgethefollowingformanyhelpfuldiscussionsandinvaluableinputinto

experimentalandequipmentdesignanddataanalysisandinterpretation:Prof.BruceMatthews,Prof

MaxHeadley,Mr.TonyMacdonald,Mr.DerekCarr,DrRogerWatkins,DrRochelleAckerleyandDrUta

Sailor.ThisworkwasfundedbytheUniversityofBristolandtheMedicalResearchCouncil(invivo

strategicskillsPhDstudentshiptoLFD).JPDisaFoulkesFoundationFellow2010.

7 Conflictofinterest

Theauthorsconfirmthattheyknowofnoconflictsofinterest.

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26

8 Figurelegends

Figure1.Behaviouraltestingapparatus.

A.Diagrammaticrepresentationofthetestingapparatuswitharatinposition.Individualthermal

modulesconsistofaPeltierdevice,heatsinkandforcetransducers.Therathindpawsarelocatedover

thesemodulesviathepositioningframe.ThetemperatureofthePeltierdeviceandtheoutputfrom

theforcetransducerisamplifiedandthencapturedviaCED’s1401whichinterfaceswithaPC.

B.Photographoftheapparatusillustratingthetwoplatesindependentlymountedonseparatethermal

modules.Inflowandoutflowtubingforcoolantfortheheatsinkscanbeseentotheleftofthe

photograph.Connectionstoamplificationandrecordingequipmentarevisibletotherightofthe

photograph.

C.PhotographofapparatusasabovebutincludingthePerspexpositioningboxandaratinsitu.

Figure2.Contactrampedheatingoftherathindpawelicitswithdrawalattemperaturesequivalentto

humandiscomfortandwithdrawalthresholds.

A.Human.Panelshowsatypicalexampleofadigitisedtraceoftherawdatageneratedfromtheforce

transducersundertheheatedindexfinger(toptrace,arbitraryunits),andthesurface/contact

temperatureofthePeltierdeviceincontactwiththefinger(lowertrace,oC),overtime(s).Detection,

discomfortandwithdrawalsareindicatedbytheverticalnumberedcursorsandthetemperatureread

fromthelowertraceasillustrated.Rateofheatingwascirca1oC/s.

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B.Rat.Panelshowsatypicalexampleofadigitisedtraceoftherawdatageneratedfromtheforce

transducersundertheheatedhindpaw(toptrace,arbitraryunits),andthesurface/contact

temperatureofthePeltierdeviceincontactwiththeplantarhindpaw(lowertrace,oC),overtime(s).

Withdrawalisindicatedbytheverticalcursor1.Temperatureisreadfromthelowertrace.Rateof

heatingwascirca1oC/s.

C.Mean(+SEM)datafromhuman(n=10)andrat(n=3)experiments.Humandetectionofheating

occursatasignificantlylowertemperaturethanratbehaviours.Humanpainthresholdandwithdrawal

thresholdarenotdifferenttotheratwithdrawaltemperature.Kruskal-Wallistest,**=p<0.05,ns=

p>0.05).

Figure3.Contactrampedcoolingoftherathindpawelicitstwodistinctbehaviouralresponses.

A.Panelshowsatypicalexampleofadigitisedtraceoftherawdatageneratedfromtheforce

transducersunderthecooledhindpaw(toptrace,arbitraryunits),andthesurface/contact

temperatureofthePeltierdeviceincontactwiththeplantarhindpaw(lowertrace,oC),overtime(s).

Therateofcoolingis-1.3oC/s.Theverticalcursorsindicatethecoolingevokedbehaviours.Cursors1,2

and3indicaterapid,transientremovalsofweightfromtheplate,andthecontacttemperatureat

whichtheyoccurred(22.1oC,19.2

oCand10.9

oC).Whenthepawwasplacedbackontheplate,there

wasoftenanincreaseinweightborneontheplate,asindicatedbytheupwardshiftofthetracemost

noticeablebetweencursors3and4.Theplatetemperaturecontinuedtofallduringthistime.Vertical

cursor4indicatesamoreprolongedremovalofweight,fullwithdrawal,at-11.87oC.

B.Scatterplotillustratingthetemperatureatwhichthetwocoolingevokedbehavioursoccurred.

HorizontalbarsshowmedianandIQRs.Themediantemperatureatwhichtheinitialtransient

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withdrawaloccurredwashigherthanthatatwhichfullwithdrawalbehavioursoccurred(p<0.001,n=10

rats,upto3rampsperrat.MannWhitneyUtest).Therateofcoolingis-1.3oC/s.

C.Scatterplotillustratingthelatenciesatwhichthetwocoolingevokedbehavioursoccurred.

HorizontalbarsshowmedianandIQRs.Themediantimetowithdrawal(latency)waslongerforfull

withdrawalsthanforinitialwithdrawals(p<0.001,n=10rats,upto3rampsperrat.MannWhitneyU

test).Therateofcoolingis-1.3oC/s.

D.Frequencyhistogramshowingthetwo-clustersolutionprovidedforresponsesvs.temperature.The

openbarsindicate“initial”withdrawals‚associatedwithclusteroneasdetectedbyhierarchical

clustering,andtheclosedbarsindicatecluster2,thefullwithdrawals.Forfullexplanationofclustering

pleaseseetext.Therateofcoolingis-1.3oC/s.

E.Frequencyhistogramshowingthetwo-clustersolutionprovidedforresponsesvs.latencies.The

openbarsindicate“initial”withdrawals‚associatedwithclusteroneasdetectedbyhierarchical

clustering,andtheclosedbarsindicatecluster2,thefullwithdrawals.Forfullexplanationofclustering

pleaseseetext.Therateofcoolingis-1.3oC/s.

Figure4.Theeffectofrateofcontactrampedcoolingonbehaviouralresponsesinrats.

A.Ascoolingrateincreased,thelatencytoinitialwithdrawaldecreased.(Median±I.Q.R,n=4rats,1-2

trialsperrate,**p<0.01,Kruskal-Wallis+Dunn’s,***p<0.001lineartestfortrendfollowinglog-log

transformation).

B.Ascoolingrateincreased,thelatencytofullwithdrawalalsodecreased.(Median±I.Q.R.,n=4rats,

1-2trialsperrate,*p<0.05,***p<0.001,Kruskal-Wallis+Dunn’s.***p<0.001lineartestfortrend

followinglog-logtransformation).

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C.Althoughthecoolingrateincreased,theplatetemperaturethatevokedinitialwithdrawalwas

unchanged,althoughthiswashighlyvariable.(Median±I.Q.R.,n=4rats,1-2trialsperrate,oneway

ANOVAwithBonferroniposthoctest).

D.Theplatetemperatureatwhichfullwithdrawalsoccurredwasalsounchangedbytherateofplate

cooling.Forfullwithdrawalstheplatetemperaturewashighlyconsistentacrosstrials.(Median±I.Q.R.,

n=4animals,1-2trialsperrateonewayANOVAwithBonferroniposthoctest).

E.Themean(±95%CI)subcutaneoustemperaturesatwhichinitialwithdrawalsoccurredatdifferent

ratesofcoolingwereinterpolatedfromthecontacttemperatures(Fig3C)andknownvaluesof

subcutaneousmeasurementsmadeinananaesthetisedrat.Unfortunately,theplatetemperaturewas

notloweredsufficientlyinthesubcutaneoustemperatureexperimenttoenabletheextrapolationof

thelowerconfidenceintervalforthe-2oC/scoolingrate.

F.Themean(±95%CI)subcutaneoustemperaturesatwhichfullwithdrawalsoccurredatdifferentrates

ofcoolingwereinterpolatedfromthecontacttemperatures(Fig.3D)asabove.

G.Platetemperatureisplottedagainstsubcutaneoustemperatureforthe4differentcoolingrates.

Therelationshipbetweenplateandsub-cutaneoustemperatureisapproximatelylinearforcooling

rates-0.5oC/sto-2

oC/s.Thesedataweregeneratedinasingleanaesthetisedrat.

Figure5.Contactrampedcoolingofthehumanforefingerallowsdefinitionofcolddetection,

discomfortandpainthresholdsandlatencies.

A.Panelshowsatypicalexampleofadigitisedtraceoftherawdatageneratedfromtheforce

transducers(arbitraryunits)underacooledforefinger(toptrace),andthesurface/contact

temperature(oC)ofthePeltierdeviceincontactwiththeforefinger(lowertrace),overtime(s).

Coolingratewas~-1.3oC/s.Participantsindicatedcolddetectionanddiscomfortandthiswasrecorded

viaafootpedal.ThisisshownintheMarkerchannelatthetopofthefigure.Theverticalcursors

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indicatetheseevents:Cursor1colddetectionat19.1oC,cursor2colddiscomfortat-2.48

oCandcursor

3coldpainandwithdrawalat-9.1oC.

B.Colddetectionthresholdsweresignificantlyhigherthatcolddiscomfortandcoldpainthresholds.

Colddiscomfortwasalsosignificantlydifferentfromcoldpain(median(I.Q.R.).***p<0.001,1way

ANOVA+Bonferroni'stest,n=10).

C.Themostcommondescriptorsusedtodescribethecoldpainevokedbycontactcoolingwere“cold”,

“numbing”and“freezing”.(n=10,2descriptorsforeachofthreetrials).

Figure6.Comparisonofhumanpsychophysicalandratbehaviouralthresholdsduringcontactramped

cooling.

A.Latencytopsychophysicalthresholdsevokedbycoolinginhumansisshownasafrequency

histogramrelativetotheplatetemperature.Thenumberofevents(leftYaxis)ineach5secondbinis

shownfordetection(dashedline),discomfort(dottedline)andwithdrawal(solidline)(n=10).Plate

temperature(oC)isshownontherightYaxis.

B.Latencytobehavioursevokedbycoolinginratsisshownasafrequencyhistogramrelativetothe

platetemperature.Thenumberofevents(leftYaxis)ineach5secondbinisshownforinitial(dashed

line)andfullwithdrawals(solidline)(n=10).Platetemperature(oC)isshownontherightYaxis.

C.Thelatenciestoinitialbriefpawremoval(rats)andcolddetection(human)wereequivalent,

althoughthevariabilityofresponseswasmuchgreaterintherats.Thelatenciesfordiscomfort,

noxiouscold(humans)andfullwithdrawals(rats)werealsonotsignificantlydifferent,againrat

responsesweremorevariable.Thelatenciesfor“initialresponse”and“pain”weresignificantly

differentinbothhumansandrats(p<0.0001).Allothercomparisonsweresignificantlydifferentexcept

forthoseindicated(KruskalWallis+Dunn’stest).BarsindicatemediansandIQRs.

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References

1. Rolke,R.,etal.,QuantitativesensorytestingintheGermanResearchNetworkonNeuropathic

Pain(DFNS):Standardizedprotocolandreferencevalues.Pain,2006.123(3):p.231-243.

2. Maier,C.,etal.,QuantitativesensorytestingintheGermanResearchNetworkonNeuropathic

Pain(DFNS):Somatosensoryabnormalitiesin1236patientswithdifferentneuropathicpain

syndromes.Pain,2010(Epubaheadofprint):p.3.

3. Cain,D.M.,S.G.Khasabov,andD.A.Simone,ResponsePropertiesofMechanoreceptorsand

NociceptorsinMouseGlabrousSkin:AnInVivoStudy.JNeurophysiol,2001.85(4):p.1561-

1574.

4. Lynn,B.andS.E.Carpenter,Primaryafferentunitsfromthehairyskinoftherathindlimb.

BrainRes,1982.238(1):p.29-43.

5. Simone,D.A.andK.C.Kajander,Excitationofratcutaneousnociceptorsbynoxiouscold.

NeurosciLett.,1996.213(1):p.53-6.

6. Simone,D.A.andK.C.Kajander,ResponsesofCutaneousA-FiberNociceptorstoNoxiousCold.

JNeurophysiol,1997.77(4):p.2049-2060.

7. Allchorne,A.J.,D.C.Broom,andC.J.Woolf,Detectionofcoldpain,coldallodyniaandcold

hyperalgesiainfreelybehavingrats.MolPain.,2005.1:p.36.

8. Walczak,J.S.andP.Beaulieu,Comparisonofthreemodelsofneuropathicpaininmiceusinga

newmethodtoassesscoldallodynia:thedoubleplatetechnique.NeurosciLett.,2006.

399(3):p.240-4.Epub2006Feb21.

9. Dhaka,A.,etal.,TRPM8IsRequiredforColdSensationinMice.Neuron,2007.54(3):p.371-

378.

10. Duraku,L.S.,etal.,RotterdamAdvancedMultiplePlate:anovelmethodtomeasurecold

hyperalgesiaandallodyniainfreelybehavingrodents.JNeurosciMethods,2014.224:p.1-

12.

11. Hargreaves,K.,etal.,Anewandsensitivemethodformeasuringthermalnociceptionin

cutaneoushyperalgesia.Pain,1988.32(1):p.77-88.

12. Jasmin,L.,etal.,Thecoldplateasatestofnociceptivebehaviors:descriptionandapplication

tothestudyofchronicneuropathicandinflammatorypainmodels.Pain.,1998.75(2-3):p.

367-82.

13. Yalcin,I.,etal.,Differentiatingthermalallodyniaandhyperalgesiausingdynamichotand

coldplateinrodents.JPain,2009.10(7):p.767-73.

14. Colburn,R.W.,etal.,AttenuatedColdSensitivityinTRPM8NullMice.Neuron,2007.54(3):p.

379-386.

15. LeBars,D.,M.Gozariu,andS.W.Cadden,Animalmodelsofnociception.PharmacolRev,

2001.53(4):p.597-652.

16. Banik,R.K.andR.A.Kabadi,AmodifiedHargreaves'methodforassessingthreshold

temperaturesforheatnociception.JNeurosciMethods,2013.219(1):p.41-51.

17. Kilkenny,C.,etal.,Improvingbioscienceresearchreporting:theARRIVEguidelinesfor

reportinganimalresearch.PLoSBiol,2010.8(6):p.e1000412.

18. McMullan,S.,D.A.A.Simpson,andB.M.Lumb,Areliablemethodforthepreferential

activationofC-orA-fibreheatnociceptors.JournalofNeuroscienceMethods,2004.138(1-

2):p.133-139.

19. Ho,H.N.andL.A.Jones,Modelingthethermalresponsesoftheskinsurfaceduringhand-

objectinteractions.JBiomechEng,2008.130(2):p.021005.

ResearchArticle

32

20. Morin,C.andM.C.Bushnell,Temporalandqualitativepropertiesofcoldpainandheatpain:

apsychophysicalstudy.Pain,1998.74(1):p.67-73.

21. Harrison,J.L.andK.D.Davis,Cold-evokedpainvarieswithskintypeandcoolingrate:a

psychophysicalstudyinhumans.Pain.,1999.83(2):p.123-35.

22. Davis,K.D.andG.E.Pope,Noxiouscoldevokesmultiplesensationswithdistincttimecourses.

Pain,2002.98(1-2):p.179-185.

23. Schepers,R.J.andM.Ringkamp,Thermoreceptorsandthermosensitiveafferents.Neurosci

BiobehavRev,2010.34(2):p.177-84.

24. Leith,J.L.,etal.,Spinalprocessingofnoxiousandinnocuouscoldinformation:differential

modulationbytheperiaqueductalgray.JNeurosci,2010.30(14):p.4933-42.

25. Mohr,C.,etal.,Centralrepresentationofcold-evokedpainreliefincapsaicininducedpain:an

event-relatedfMRIstudy.Pain,2008.139(2):p.416-30.

26. Kauppila,T.,V.K.Kontinen,andA.Pertovaara,Influenceofspinalizationonspinal

withdrawalreflexresponsesvariesdependingonthesubmodalityoftheteststimulusandthe

experimentalpathophysiologicalconditionintherat.BrainRes,1998.797(2):p.234-42.

27. Meyer,R.A.,etal.,PeripheralMechanismsofCutaneousNociception,inWallandMelzacks

TextbookofPain,S.B.McMahonandM.Koltzenburg,Editors.2006,ChurchillLivingstone.p.

3-34.

28. Tillman,D.B.,etal.,ResponseofCfibrenociceptorsintheanaesthetizedmonkeytoheat

stimuli:correlationwithpainthresholdinhumans.JPhysiol,1995.485(Pt3):p.767-74.

29. Tabo,E.,J.H.Eisele,Jr.,andE.Carstens,Forceoflimbwithdrawalselicitedbygradednoxious

heatcomparedwithotherbehavioralmeasuresofcarrageenan-inducedhyperalgesiaand

allodynia.JNeurosciMethods,1998.81(1-2):p.139-49.

30. Brown,R.T.andJ.G.Baust,Timecourseofperipheralheterothermyinahomeotherm.AmJ

Physiol,1980.239(1):p.R126-9.

31. Gordon,C.J.,Thermalbiologyofthelaboratoryrat.PhysiolBehav,1990.47(5):p.963-91.

32. Sherrington,C.,Theintegrativeactionofthenervoussystem.1947,Cambridge:Cambridge

UniversityPress.

33. Dunham,J.P.,etal.,TransientreceptorpotentialchannelA1andnoxiouscoldresponsesinrat

cutaneousnociceptors.Neuroscience,2010.165(4):p.1412-9.

34. Beise,R.D.,E.Carstens,andL.U.Kohlloffel,Psychophysicalstudyofstingingpainevokedby

brieffreezingofsuperficialskinandensuingshort-lastingchangesinsensationsofcooland

coldpain.Pain,1998.74(2-3):p.275-86.

35. Lenhardt,R.,Theeffectofanesthesiaonbodytemperaturecontrol.FrontBiosci(ScholEd),

2010.2:p.1145-54.

36. Davis,K.D.,Cold-inducedpainandprickleintheglabrousandhairyskin.Pain,1998.75(1):

p.47-57.

37. Yarnitsky,D.andJ.L.Ochoa,Releaseofcold-inducedburningpainbyblockofcold-specific

afferentinput.Brain,1990.113(Pt4):p.893-902.

38. Johnson,K.O.,I.Darian-Smith,andC.LaMotte,Peripheralneuraldeterminantsof

temperaturediscriminationinman:acorrelativestudyofresponsestocoolingskin.J

Neurophysiol,1973.36(2):p.347-70.

39. Rief,W.andJ.A.Glombiewski,Thehiddeneffectsofblinded,placebo-controlledrandomized

trials:anexperimentalinvestigation.Pain,2012.153(12):p.2473-7.

40. Kotsis,V.,etal.,Perceivedtreatmentgroupaffectsbehavioralandneuralresponsesto

visceralpaininadeceptiveplacebostudy.NeurogastroenterolMotil,2012.24(10):p.935-

e462.

41. Defrin,R.,etal.,Spatialsummationandspatialdiscriminationofcoldpain:effectofspatial

configurationandskintype.Pain,2011.152(12):p.2739-45.

ResearchArticle

33

42. Leem,J.W.,W.D.Willis,andJ.M.Chung,Cutaneoussensoryreceptorsintheratfoot.J

Neurophysiol,1993.69(5):p.1684-99.

43. Kress,M.,etal.,Responsivenessandfunctionalattributesofelectricallylocalizedterminalsof

cutaneousC-fibersinvivoandinvitro.JNeurophysiol,1992.68(2):p.581-595.

44. Teliban,A.,etal.,Responsesofintactandinjuredsuralnervefiberstocoolingandmenthol.J

Neurophysiol,2014.111(10):p.2071-83.

45. Darian-Smith,I.,K.O.Johnson,andR.Dykes,"Cold"fiberpopulationinnervatingpalmarand

digitalskinofthemonkey:responsestocoolingpulses.JNeurophysiol,1973.36(2):p.325-

346.

46. Adriaensen,H.,etal.,Responsepropertiesofthinmyelinated(A-delta)fibersinhumanskin

nerves.JNeurophysiol,1983.49(1):p.111-22.

47. Campero,M.,etal.,Slowlyconductingafferentsactivatedbyinnocuouslowtemperaturein

humanskin.JPhysiol.,2001.535(Pt3):p.855-65.

48. Klement,W.andJ.O.Arndt,Painbutnotemperaturesensationsareevokedbythermal

stimulationofcutaneousveinsinman.NeurosciLett,1991.123(1):p.119-22.

49. Klement,W.andJ.O.Arndt,Theroleofnociceptorsofcutaneousveinsinthemediationofcold

paininman.JPhysiol,1992.449:p.73-83.

50. Craig,A.D.,Aratisnotamonkeyisnotahuman:commentonMogil(NatureRev.Neurosci.

10,283-294(2009)).NatRevNeurosci,2009.10(6):p.466.

A

B C

marker

7 8 9

49.13oC 50.9oC

39.62oC

Fo

rce

(a

.u)

Te

mp

(oC

)

3

2.5

2

1.5

50

40

30

Fo

rce

(a

.u)

Te

mp

(oC

)

50

40

30

20

4

3

2

748 758 768

478 488 498

46.32oC

1

A

B

C

Fo

rce

(a

.u)

Te

mp

(oC

)

40

20

4

3

2

0

10.93oC

19.21oC 22.11oC

-11.87oC

1 2 3

4

5

265 275 285 295 \ 

A

B

C

D

A B

C D

E F

G

A

B

Fo

rce

(a

.u)

Te

mp

(oC

)

1 2 3 3.0

2.5

C

marker 3.5

19.1oC

-2.48oC

-9.1oC

20

-20

0

770 780 790 800 810

A

B

C

Eve

nts

E

ve

nts

20

0

15

10

5

0 5 10 15 20 25 30 35 40 45 50

Te

mp

era

ture

(oC

)

0

10

-10

-20

20

30 E

ve

nts

20

0

15

10

5

0 5 10 15 20 25 30 35 40 45 50

Te

mp

era

ture

(oC

)

0

10

-10

-20

20

30

Latency (s)

Latency (s)

humans

rats