Single-trial averaging improves the physiological interpretation of contact heat evoked potentials.

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ContentslistsavailableatScienceDirect

NeuroImage

journalhomepage:www.elsevier.com/locate/neuroimage

Single-trial averaging improves the physiological interpretation of contact heat evoked potentials

Catherine R. Jutzeler

^a^,^b^,^c^,¹^,^∗

, Lukas D. Linde

^d^,^e^,^f^,¹

, Jan Rosner

^c^,^g

, Michèle Hubli

^c

, Armin Curt

^c

, John L.K. Kramer

^d^,^e^,^f^,^∗

aSwiss Federal Institute of Technology (ETH Zurich), Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058 Basel, Switzerland

bSIB Swiss Institute of Bioinformatics, Switzerland

cSpinal Cord Injury Center, University Hospital Balgrist, University of Zurich, Zurich, Switzerland

dICORD, University of British Columbia, 818W 10th Ave, Vancouver, British Columbia, Canada

eDepartment of Anesthesiology, Pharmacology & Therapeutics, Faculty of Medicine, University of British Columbia, 818W 10th Ave, Vancouver, British Columbia, Canada

fDjavad Mowafaghian Centre for Brain Health, University of British Columbia, 818W 10th Ave, Vancouver, British Columbia, Canada

gDepartment of Neurology, University Hospital Bern, Inselspital, University of Bern, Bern, Switzerland

a r t i c le i n f o

Keywords:

Contact heat Evoked potentials Ageing

Single-trial averaging Pain

a b s t r a ct

Laserandcontactheatevokedpotentials(LEPsandCHEPs,respectively)provideanobjectivemeasureofpath- waysandprocessesinvolvedinnociception.ThemajorityofstudiesanalyzingLEPorCHEPoutcomeshavedone sobasedonconventional,across-trialaveraging.Withthisapproach,evokedpotentialcomponentsarepotentially confoundedbylatencyjitterandignorerelevantinformationcontainedwithinsingletrials.Thecurrentstudy addressedtheadvantageofanalyzingnociceptiveevokedpotentialsbasedonresponsestonoxiousstimulations withineachindividualtrial.Single-trialandconventionalaveragingwereappliedtodatapreviouslycollectedin 90healthysubjectsfrom3stimulationlocationsontheupperlimb.Theprimaryanalysisfocusedonrelation- shipsbetweensingleandacross-trialaveragedCHEPoutcomes(i.e.,N2P2amplitudeandN2andP2latencies) andsubjectcharacteristics(i.e.,age,sex,height,andratingofperceivedintensity),whichwereexaminedby wayoflinearmixedmodelanalysis.Single-trialaveragingleadtolargerN2P2amplitudesandlongerN2and P2latencies.Ageandratingsofperceivedintensityweretheonlysubjectlevelcharacteristicsassociatedwith CHEPsoutcomesthatsigniﬁcantlyinteractedwiththemethodofanalysis(conventionalvssingle-trialaveraging).

Thestrengthofrelationshipsforageandratingsofperceivedintensity,measuredbylinearﬁt,wereincreased forsingle-trialcomparedtoconventionalacross-trialaveragedCHEPoutcomes.Byaccountingforlatencyjit- ter,single-trialaveragingimprovedtheassociationsbetweenCHEPsandphysiologicaloutcomesandshouldbe incorporatedasastandardanalyticaltechniqueinfuturestudies.

1. Introduction

Contactheatandlaserevokedpotentials(CHEPsandLEPs,respectively)representrecruitmentofthinlymyelinatedA-deltaﬁbresinthe periphery,conductionin thespinothalamic tract, andareassociated withtheperceptionofpain(Chenetal., 2001;Haefelietal.,2013b; Jutzeler etal., 2016; Krameret al., 2009). While neuralactivity of CHEPsandLEPs maynot directly reﬂect centralprocessing of noci- ceptionandpain(MourauxandIannetti,2018,2009),bothmeasures arereliablyemployedtoassessthefunctionofthespinothalamicpath- waysandpain perception (Chen etal., 2001; Haefeli et al., 2013a;

∗Correspondingauthors.

E-mailaddresses:Catherine.Jutzeler@bsse.ethz.ch(C.R.Jutzeler),kramer@icord.org(J.L.K.Kramer).

1 Theseauthorscontributedequallytothiswork.

Jutzeleretal.,2016;Krameretal.,2012b)withinsubjects(HuandIan- netti,2019).

The amplitudes and latencies of nociceptive evoked potentials are dependent on numerous factors, including stimulation location (Granovskyetal.,2005;Haefelietal.,2013b),age(Chaoetal.,2007; Granovsky et al., 2016;Jutzeler et al., 2016),andsex (Chen etal., 2006;deTommasoetal.,2017;Granovskyetal.,2016;Staikouetal., 2016; Truini et al., 2005). Location related variability is primar- ily attributable to diﬀerences in peripheral conduction distances (Magerland Treede, 1996; Truiniet al., 2005),temporaldispersion (Iannettietal.,2006;Krameretal.,2013),andreceptordensitygra- dients(Athertonetal.,2007;Perrettietal.,2003;Ragé etal.,2011).In

https://doi.org/10.1016/j.neuroimage.2020.117473

Received2June2020;Receivedinrevisedform12September2020;Accepted14October2020 Availableonline21October2020

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addition,behavioral relevanceofstimuluslocationfurtherinfluences waveformsparameters of evoked potentials, asmore proximal loca- tions tend toresult in larger and earlier responses (Bufacchi et al., 2016;BufacchiandIannetti,2018;Samboetal.,2012;SamboandIan- netti,2013).Microstructuralchangeswithinthesomatosensorynervous systemassociatedwithaginggiverisetoloweramplitudeandlonger latency ofnociceptive evokedpotentials in olderadults (Jacobs and Love, 1985; Lauriaet al., 1999). Sex differences,while variably reported(Chenet al., 2006; deTommaso et al., 2017; Staikouet al., 2016; Truini et al., 2005), tend to provide objective evidence that womenare more sensitivetonoxious stimulation compared tomen (Granovskyetal.,2016),evenafteradjustingforrelevantsubjectchar- acteristics(e.g.,height)(Jutzeleretal.,2016).Collectively,thesefind- ingsdemonstratetheinherentvalueofCHEPsandLEPstodepictbiolog- icallyrelevantinformationunderlyingnoxiousheatstimulationapplied intheperiphery.

ThestandardacquisitionofeitherCHEPsorLEPsinvolvestherepet- itiveapplicationofnoxiousstimuliatlonginter-stimulusintervals,from whichwaveformsareconventionallyaveragedforvisualinspectionand evaluation(Chenetal.,2006;Krameretal.,2013).Whileeﬀectivein increasingsignaltonoiseratios,amajordisadvantageofthisapproach isthattrialspeciﬁcvariationsinamplitude,latency,andmorphologyof evokedpotentialswithinasubject,betweenstimuliareminimizedand distortedfromacrosstrialaveraging(MourauxandIannetti,2008).An importantexampleofsuchinter-trialvariabilityislatencyjitter,which describesthevariationinlatenciesofN2andP2waveforms.Thecon- ventionalanalysisofacrosstrialaveraging,withoutaccountingforla- tencyjitter,candistortevokedpotentialcomponents,andthusamajor concernisthatthispracticeremovesbiologicallyrelevantinformation.

Asanalternative,singletrialaveragingtechniqueshavebeendeveloped toextract waveformcharacteristicsfromevokedpotentialsgenerated inresponsetoindividualstimuli(Hatemetal.,2012;Huetal.,2011, 2010;Huangetal.,2013;Mayhewetal.,2006;Warbricketal.,2009).

SingletrialaveragingtendstoincreaseLEPamplitudes(Huetal.,2011; Warbricketal.,2009),improvingtheclarityofevokedpotentialwave- formsbyaccountingfortrialtotrialvariations.Despitetheseadvances insignalprocessing(MourauxandIannetti,2008),relativelyfewstud- ieshaveadoptedsingletrialapproachtoanalyzeandinterpretCHEPs (Warbricketal.,2009).

The present study addressed the hypothesis that the single-trial compared to across trial averaging approach would better capture physiologicalrelevantinformationinresponsestonoxiousstimulation, speciﬁcallybyaccountingforlatencyjitterinN2P2waveforms.This inturnwillstrengtheningtherelationshipsbetweenCHEPoutcomes, subjectcharacteristics,andstimuluslocation.Single-trialanalysiswas performedon previously publishedCHEPsdata using anestablished technique(Hatemetal.,2012;Huetal.,2011,2010)andcomparedto outcomesfromconventionalaveraging(Jutzeleretal.,2016).

2. Methods

Toaddressourhypothesis,weutilizedalargeCHEPsdatasetprevi- ouslypublishedbyJutzeleretal.,2016(Jutzeleretal.,2016).Thisstudy focusedonnormativeCHEPoutcomes,basedonconventionalaveraging only.Forcomparativepurposes,theresultsofconventionalaveraging areincludedhereagain.All procedureswerein accordancewiththe DeclarationofHelsinki andapprovedbythelocalethicsboard‘Kan- tonaleEthikkommissionZurich,KEK’(ref.number:EK-04/2006,cini- caltrial.govnumber:NCT02138344).Participantsprovidedwrittenin- formedconsent.

2.1. Subjects

Onehundredandﬁveneurologicallyhealthysubjectswererecruited throughonlineandprintedadvertisements.Inclusioncriteriacomprised ageof18–80yearsandnativelanguagebeingeitherEnglishorGerman.

Exclusioncriteriaincludedpregnancy,intakeofanymedication(except birthcontrol),andanyobviousneurologicalcondition.

2.2. Studyprotocol

CHEPs were recorded afterthermally stimulatingthe C4 (shoulder),C6(baseofthethumb),andC8dermatomes(baseofdigitusmin- imus).Normalbaselinestimulations(35°Cbaseline,rampedtoapeak temperatureof52 °C,atarateof 70°C/s) wereemployedtorecord CHEPs,describedpreviously(Haefelietal.,2013a;Jutzeleretal.,2015; Krameretal.,2012b).DuringtheacquisitionofCHEPs,subjectswere lyinginasupinepositionwitheyesopen.Inordertominimize ocu- larartefacts,subjectswereinstructedtofocusonapointontheceil- ing,minimizeblinking,aswellastoremainrelaxedandquietduring testing.Traces contaminatedwithmuscleorblinkartefacts wereex- cluded inrealtimeandadditionalstimuliwereappliedtorecord15 artefactfreetracesperlocation.Contactheatstimuliwereappliedwith aninter-stimulustimeintervalthatrandomlyvariedbetween8and12s (Haefelietal.,2013a;Jutzeleretal.,2015;Krameretal.,2012b).Cued byanauditorysignaltwosecondspoststimulus,subjectswereinstructed toratetheperceivedpainofeachstimulusfrom0(nopain)to10(most unbearablepain).Theauditorycuealsoprovided anopportunityfor participantstoblinkandtherefore,avoidblinkingduringheatstimula- tions.TheCHEPsthermodewasslightlyrepositionedaftereachstimulus withinthedermatometestedtoreducereceptorfatigueorsensitization byoverheatingtheskin(Granovskyetal.,2005).

2.3. Stimulatingdeviceandrecordingsetup

Acontactheatstimulatorwasemployedtodeliverstimulation(Path- way,Medoc,RamatYishai,Israel).TheCHEPthermodesurface(diam- eter:27mm)consistsofaheatingthermo-foilcoveredwithalayerof thermo-conductiveplastic.Thenominalheatingrateofthisdeviceis 70°C/s,withacoolingrateof40°C/s.

Corticalresponsestothenoxiousheatwererecordedwith9mm Ag/AgCl surfacediskelectrodes ﬁlled withconductive adhesivegel.

ScalprecordingsiteswerepreparedwithNuprep(D.O.Weaver&Co.

Aurora,CO)andalcohol.Electrodeswerepositionedinaccordancewith theInternational10–20system.BothN2 andP2wereacquiredfrom anactivevertexrecordingelectrode(Cz)referencedtolinkedearlobes (A1-A2).Therationaleforareducedelectrodesetup arosefrom the factthatconsistentnegativeandpositivepotentials,labelledN2andP2, arereliablydetectedatCz(Chenetal.,2006;Granovskyetal.,2016; Haefelietal.,2013a;Jutzeleretal.,2015;Krameretal.,2012b).All signalsweresampledat2000Hzusingapreampliﬁer(20000x,band- passﬁlter1-300Hz,ALEASolutions,Zurich,Switzerland).Datawere recordedwith100mspre-triggerandaonesecondpost-triggerinacus- tomizedLabView(V1.43CHEP,ALEASolutions,Zurich,Switzerland) program.

2.4. Conventionalaveraging

FilteredCHEPsfrom15 artefactfree trialswereaveragedandvi- sually inspected forN2 and P2waveforms. Toensureadequate sig- naltonoiseratioforthedeterminationofwaveformparameters,aver- agedamplitudesoflessthan10uVwereexcludedfromfurtheranalysis (Jutzeleretal.,2016).

2.5. Singletrialanalysis

Singletrialanalysiswasperformedusinganopenlyavailablepro- gram (Huetal.,2011, 2010).Inbrief,singletrialaveragingusinga combinationofwaveletﬁlteringandmultiplelinearregressionstode- terminewaveformparameters(N2/P2amplitudesandlatencies)from individualtrials(Huetal.,2011,2010).Waveletﬁlteringwasemployed

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Fig.1. Representativetracesofconventionalaveragingandsingletrialaveraginganalysismethods.A)Individualcontactheatevokedpotential(CHEP)waveforms, ﬁlteredandre-referenced.B)ConventionalaveragingofindividualCHEPwaveforms,fromwhichN2andP2outcomearederived.C)IndividualCHEPwaveforms followingsingletrialanalysis,viawaveletﬁlteringandmultiplelinearregressionwithdispersionterm(Huetal.,2011,2010).D)AveragedCHEPoutcomes determinedfromsingletrialanalysis.

toenhancethesignaltonoiseratioandfacilitatetheestimationofla- tencyandamplitudeofsingletrialevokedpotentialpeaks(Huetal., 2011,2010;Krameretal.,2013).Toperformanunbiasedsingletrial analysis,anautomatedapproachusingmultiplelinearregressionwith adispersionterm(MLRd)isthenimplemented(Huetal.,2011).The dispersiontermenhancestheabilityofmultiplelinearregressionsto detectchangesinwaveformmorphologyandprovidesamoreaccurate measureoflatencyandamplitudeofsingletrialevokedpotentialpeaks (Huetal.,2011).Waveformpeakswereautomaticallydetectedwithin a100-millisecondwindowofthewavelet-ﬁlteredaverageN2andP2.

IndividualtrialN2andP2waveformparameterswerethenaveraged together,toprovidethesingletrialanalysisoutcomesforeachpartici- pant(Fig.1).

2.6. Statisticalanalysis

CHEPoutcomesfromconventionalaveragingandsingle-trialaver- agingwereexaminedusinglinearmixedeffectsmodels.ForeachCHEP outcome(N2P2amplitude,N2latency,P2latency),fixedeffectsofder- matome(C4,C6,C8),andanalysismethod(conventionalvssingletrial averaging)wereassessedinalinearmixedmodel,withrandomeffects ofparticipants.Subjectcharacteristics(age,sex,height,andratingof perceivedintensity) wereincludedin these linearmixed models,ex- aminingtheoveralleffectofanalysismethodanddermatomeonCHEP outcomes.Subsequentmodelswithaninteractiontermbetweeneach subjectcharacteristicandanalysismethodwereincludedtoexploreof influenceofanalysismethodontherelationshipsbetweenCHEPout- comesandsubjectcharacteristics.Significantinteractionswerefollowed

upvialinearmixedmodelsforeachanalysismethod,todeterminere- spectivediﬀerencesinCHEPoutcomesandtherelationshiptosubject characteristicsbetweenconventionalandsingle-trialaveraging.Based onpreliminarylinearmixedmodelanalysis,multiplelinearregressions between age,ratingofperceivedintensity,andCHEPoutcomeswere furtherexploredforbothanalysismethods.Bonferronicorrectionwas appliedtoadjustformultiplecomparisons.Analphalevelof0.05was usedforallstatisticaltests.RStatisticalSoftware(version3.5.3,MacOS 10.14.6Mojave)wasusedforallstatisticalanalysesandproducingall plots(RCoreTeam,2019;Wickham,2016).

2.7. Simulateddataanalysis

Tofurtherexploretheeffectsofconventionalaveragingandsingle trial analysis onCHEPs outcomes,we performed a smallsimulation experiment. The goal here was to systematically manipulate the amplitudes andlatencies of individual trialwaveformsto determine the comparative effects on conventional averaging and single trial analysis, respectively. A single waveform was artificially increased in amplitudeby 40%,andthelatency wasshifted20mstotheleft.

Thus,twowaveforms(smallandlarge)wereusedforoursimulation.

First,thesmallwaveformwasreplicated,suchthata15-trialdataset contained only thesmall waveform. Then, a single large waveform trialwas added,withasmallwaveformremoved. Thiswas repeated untilonlylargewaveformsremained.Foreachdataset,weperformed conventional (across-trial) averaging and single trial analysis, and N2P2 outcomes were compared for each dataset. Findings from the simulationandexampletracesofwaveformscanbefoundinFig.7.

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Fig.2. Contactheatevokedpotentials(CHEP)N2P2am- plitudes(A),N2latencies(B),andP2latencies(C)from cervicalspinedermatomes(C4,C6,C8).Conventionalav- eraging(CV) andsingletrialaveraging(STA)analysis methodscomparedwithineachdermatome/CHEPout- comes.Agegroupsseparatedintoyoung(18–40yrs),mid- dle(41–60yrs),andelderly(61–80yrs).Lettersdenote significantdifferencesbetweendermatomesforbothstim- ulationprotocols,suchthatdifferentletterscorrespondto significantdifferencesbetweendermatomes.ForpanelB specifically,differentlettersdenotesignificantdifferences betweendermatomesandbetweenanalysismethods(i.e.

aissignificantlydifferentfromb,c,cd,andc;dissignifi- cantlydifferentfromc,butneitheraresignificantlydiffer- entfromcd).Linearmixedmodelswereadjustedforage, sex,andheight,withasignificancelevelofalpha<0.05.

3. Results

3.1. Cohortsummary

Out of 105 neurologically healthy participants, four were previ- ouslyexcludedduetointolerancetoheatstimuliandpoordataquality (Jutzeleretal.,2016).Anadditionaleightparticipantswereexcluded fromthepresentanalysisduetopoordataquality(i.e.,conventional averagedN2P2amplitudesbelow10uVorsubstantialartefactsthatin- ﬂuencedsingletrialanalysis).Theremaining93individualscomposed of45menand48womenwithameanage45.8±16.8years(range:

19–80years)wereincludedinthebothanalysismethods(conventional averagingandsingletrialanalysis).Individualcharacteristicsaresum-

Table1

Participantcharacteristics(mean±standarddeviation).

Sex Males ( N = 45) Females ( N = 48) Age (years) 44.2 ± 15.4 47.2 ± 18.0 Height (cm) 168.1 ± 7.0 178.4 ± 7.1

marizedinTable1.SummaryN2P2amplitude,N2latency,andP2la- tencyfollowingconventionalandsingle-trialaveragingaresummarized inTable2.RepresentativeCHEPs(N2/P2)individualtrialtracesofcon- ventionalandsingle-trialaveragingareillustratedinFig.1.

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Table2

Contactheatevokedpotential(CHEP)summaryoutcomes(mean±standarddeviation).

CHEP Outcome Analysis Method

Dermatome

C4 C6 C8

N2P2 Amplitude (uV) CA 27.0 ± 11.7 23.8 ± 7.4 23.2 ± 8.5

STA 28.2 ± 12.2 25.2 ± 9.8 24.1 ± 11.4

N2 Latency (ms) CA 361.4 ± 31.3 383.6 ± 31.1 402.3 ± 31.6 STA 380.8 ± 37.9 414.6 ± 41.2 420.3 ± 53.8 P2 Latency (ms) CA 504.4 ± 44.2 527.5 ± 61.2 530.0 ± 46.5 STA 513.9 ± 43.8 536.8 ± 53.6 541.5 ± 62.4 CA-Conventionalaveraging.

STA– Single-trialaveraging.

Fig.3.LinearregressionsbetweenagingandN2P2amplitudefor eachdermatome(C4,C6,C8)andanalysismethod(conventional averagingandsingletrialaveraging).Strengthoflinearregression (R²)isgivenforeachdermatome,acrossanalysismethods.

3.2. Maineﬀectsofanalysismethod

ThereweresigniﬁcantmaineﬀectsofanalysismethodonN2P2am- plitude(F_(1408.3)=5.97,p<0.05),N2latency(F_(1413.7)=69.8,p<0.001), orP2latency(F_(1410.3)=6.37,p<0.05).Overall,single-trialaveraging resultedinlargerN2P2amplitudesandlongerN2andP2latencies.

3.3. Maineﬀectsofstimuluslocation

Therewere signiﬁcantmaineﬀectsof stimuluslocationon N2P2 amplitude(F_(2420.7)=9.92,p<0.001), andboth P2(F_(2428.7) =72.7,

p<0.001)andN2latencies(F_(2422.4)=20.0,p<0.001).Generally,laten- cieswerelonger,andamplitudesweresmallerinC6andC8(i.e.,hand) dermatomescomparedtoC4(i.e.,shoulder).Speciﬁccomparisonsbe- tweendermatomesforCHEPoutcomesarefurtheroutlinedinFig.2. 3.4. Maineﬀectsofsubjectcharacteristics

There was a significant main effect of age on N2P2 amplitude (F_(1,87.4) =18.4,p<0.001),N2latency(F_(1,86.3) =4.72,p<0.05),and P2latency(F_(1,83.0)=8.00,p<0.01).Ouranalysisalsorevealedasig- nificant maineffect of sexon N2 (F_(1,87.5) = 7.06, p<0.01) and P2

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Fig.4.LinearregressionsbetweenagingandN2latencyforeach dermatome(C4,C6,C8)andanalysismethod(conventionalaver- agingandsingletrialaveraging).Strengthoflinearregression(R²) isgivenforeachdermatome,acrossanalysismethods.

latencies(F_(1,85.6)=10.32,p<0.01),aswellasamaineﬀectofrating ofperceivedintensityonN2P2amplitude(F_(1480.6)=31.7,p<0.001).

NosigniﬁcantmaineﬀectofheightonanyCHEPoutcomeswasfound (N2P2:F_(1,85.8)= 0.29,p= 0.59;N2:F_(1,84.0)= 1.23,p= 0.27;P2:

F_(1,81.4)=0.01,p=0.91).

3.5. Interactioneﬀectsbetweenmethodofanalysis,stimulationlocation, andsubjectcharacteristics

There was a significant age by analysis method interaction effect for N2P2 amplitude (F_(1408.0) = 4.60, p<0.05) and P2 latency (F_(1407.1) = 8.55, p<0.01). These interactions suggest that the relationship between age and CHEP outcomes depends on the method ofanalysis.Linearmixedmodelsperformedseparatelyforsingle-trial andconventionallyaveragedCHEPsrevealedsignificantmaineffects of agefor N2P2amplitudeforboth analysismethods (conventional:

F_(1,87.0)=13.7,p<0.001;singletrial:F_(1,86.5)=21.8,p<0.001),whilethe effectofagewassignificantduringsingletrialaveragingonlyforP2la- tency(conventional:F_(1,86.4)=1.16,p=0.28;singletrial:F_(1,81.8)=16.5, p<0.001).ForbothN2P2amplitudeandP2latency,thebetacoefficients foragefromlinearmodelswerehigherforsingle-trialaveraging(N2P2:

ß=−0.31,t=−4.8,p<0.001;P2:ß=0.84,t=2.5,p<0.05)compared toconventional averaging(N2P2:ß=−0.25,t=−4.3,p<0.001;P2:

ß=0.27,t=0.83,p=0.40).Collectively,theseobservationssuggest

thattherelationshipbetweenageandCHEPsisstrengthenedbysingle- trialanalysis.SpeciﬁclinearrelationshipsbetweenagingandCHEPout- comesforeachdermatomeandanalysismethodarefurtheroutlinedin Figs.3–5,whiletherelationshipsbetweenratingofperceivedintensity andN2P2amplitudeareoutlinedinFig.6.

There was a significant ratingof perceived intensityby analysis methodinteractionforN2P2amplitude(F_(1411.7)=4.24,p<0.05).This suggeststhattherelationshipbetween N2P2amplitudeandratingof perceived intensitydepends onthemethod ofanalysis. Separatelin- earmixedmodelsrevealsignificantmaineffectsofratingofperceived intensity on N2P2 amplitudefor conventional andsingle trial aver- agedanalysismethods(conventional:F_(1250.3)=10.0,p<0.001;single trial: F_(1231.7) = 15.9,p<0.001).The betacoefficients ratingof per- ceivedintensityfromlinearmodelswashigherforsingletrialaverag- ing(ß=1.74,t=3.53,p<0.001)comparedtoconventionalaveraging (ß=1.55,t=3.70,p<0.001).Similartoaging,thesecollectiveobser- vationssuggestthattherelationshipbetweenratingofperceivedinten- sityandN2P2amplitudeisstrengthenedbysingletrialanalysis.There werenosignificantinteractionsforsexorheightandanalysismethod foranyCHEPoutcomes.Therewerealsonothree-wayinteractionsbe- tweensubjectcharacteristics,analysis,anddermatomeforanyCHEP outcomes.

Therewasnosigniﬁcantinteractionbetweendermatomeandanal- ysisforN2latency(F_(2406.4)=2.59,p=0.08).Pairwisecomparisons

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Fig.5.LinearregressionsbetweenagingandP2latencyforeach dermatome(C4,C6,C8)andanalysismethod(conventionalaver- agingandsingletrialaveraging).Strengthoflinearregression(R²) isgivenforeachdermatome,acrossanalysismethods.

between analysis methods revealedsigniﬁcantly longerN2 latencies forsingletrialaveragingcomparedtoconventional averagingforC4 (t=−8.4,p<0.001,Cohen’sd=0.56),C6(t=−11.8,p<0.001,Cohen’s d=0.85),andC8(t=−4.0,p<0.001,Cohen’sd=0.44)dermatomes.

Separatelinearmodelscalculatedforconventionalaveragingandsin- gletrialaveragingrevealeddifferencesbetweendermatomes.Forsingle trialaveraging,N2latenciesforC6(t=5.8,p<0.001)andC8(t=6.5, p<0.001)weresignificantlylongerthanC4,withnosignificantdiffer- encesbetweenC6andC8(t=1.06,p=0.29).Thisfitswithincreased peripheralconductiondistanceassociatedwithstimulatingtheshoul- dercomparedtostimulatingthehand.Conventionalaveragingdemon- stratedsignificantlylongerN2latenciesforC6(t=9.2,p<0.001)and C8(t=16.5,p<0.001)comparedtoC4,andalsodemonstratedafur- thersignificantincreaseforN2latenciesinC8comparedtoC6(t=8.4, p<0.001)(Fig.2).

3.6. Simulateddataﬁndings

N2latencieswerelongerwithsingletrialanalysiscomparedtocon- ventional(across-trial) averagingwhenalow numberoflargewave- formswereincludedindatasets(Fig.7A).Asprogressivelymorelarge waveformswereincluded,conventionalaveragingandsingletrialanal- ysis N2 latenciesconverged(Fig.7A).Asimilar trend was observed forP2latencies,albeitwithlessconvergencewithprogressivelymore

largeamplitudewaveforms(Fig.7B).N2P2amplitudeweremostconsis- tentbetweenanalysismethodswithsimilarnumberofsmallandlarge amplitudetrials,whileextremespresented thelargest diﬀerencesbe- tweenanalysismethods(Fig.7C).Overall,conventionalaveragingand singletrialanalysisaresimilarweresimilarwhendatasetscontained mostlylargewaveformswithafewsmallwaveforms.Conversely,anal- ysismethodsdeviatedsubstantiallywhendatasetscontainedafewlarge amplitudetrials.

4. Discussion

Inthecurrentstudy,single-trialaveragingleadtolargeramplitude CHEPswaveformsandlongerlatencies.Moreover,thesesubtlechanges in N2P2waveformssigniﬁcantlychanged theinterpretation ofCHEP outcomes comparedtoconventional(across-trialaveraging) analysis.

Single trial averaging revealed strongerassociations with peripheral conductiondistances,age,andratingofperceivedintensitycompared toconventionalaveraging.Overall,ourresultsdemonstratetheadvan- tageofsingletrialaveragingtocapturebiologicallyrelevantinformation pertainingtotheacquisitionofCHEPs.

The applicationof singletrial analysistonociceptive evokedpo- tentialsdatesbackmorethan25years,toseminalresearchexploring time-shifted averagingof LEPs(Purves andBoyd,1993).Subsequent approachesutilizedwavelettransformationasameansofﬁlteringindi-

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Fig.6. LinearregressionsbetweenpainratingandN2P2ampli- tudeforeachdermatome(C4,C6,C8)andanalysismethod(con- ventionalaveragingandsingletrialaveraging).Strengthoflinear regression(R²)isgivenforeachdermatome,acrossanalysismethods.

vidualevokedpotentials(MourauxandPlaghki,2004)andincorporated automatedpeakdetectionbymultiplelinearregression(Mayhewetal., 2006),beforeultimatelyarrivingatthemethodemployedinthecur- rentstudy(Huetal.,2011,2010).Whiledemonstratingthatsingletrial analysisisclearlypossible,decidedlymissing,tothispoint,hasbeen evidencethatsingletrialanalysisimprovesthedetectionofbiologically relevantaspectsofnociception.Thelackofthisknowledgehaslikely,in part,contributedtolimiteduptakeamongresearchers,forwhomsingle trialanalysiscomesatthecostofincreasedanalysistimeandgreater complexitycomparedtoconventionalaveraging.

Towardsshiftingthediscussionfromtheorytopractice,ourresults demonstratetheinherentvalueofsingletrialanalysisfornociceptive evokedpotentials.Thiswasevidencedbythreekeyobservations.The first is that single trial analysis nullified N2 latency differences be- tweenC6andC8stimulationsites,readilyapparentwithconventional averaging. Wehave reportedsignificant differences between C6and C8stimulationsitespreviouslybasedonananalysisofthesamedata (Jutzeleretal.,2016)andalsoobservedtrendstowardssimilardiffer- encesin other,independentdatasets thatincorporatingconventional averaging(Haefelietal.,2013b).Basedonmatching peripheralcon- ductiondistancesandmarginaldifferencescentrally,C6(i.e.,baseof thethumb) andC8 (i.e.,base of the4th finger)stimulationshould, fromaneurophysiologicalperspective,yieldsimilarlatencies.Theres- olutionofthisdiscrepancybysingletrialanalysissuggeststhatdiffer-

encesbetweenC6andC8,asreportedpreviously(Haefelietal.,2013b; Jutzeleretal.,2016),areanartefactofconventionalaveraging.Specif- ically,stimulationoftheC8dermatomeatthebaseofthe5thﬁnger mayyieldmorelatencyjitterduetoalessevenlydistributedactiva- tionofcutaneousthermo-nociceptors,owingtotheanatomicalstructure oftheskinareaandsizeoftheheatstimulator.Thesechallengesmay alsoresultinunintentionalstimulationofglabrousskin,knowntoresult inlongerCHEPslatencies(Hüllemannetal.,2019).Thedevelopment ofsmaller,moreeﬀectivecontactheatstimulationdevices(DeKeyser etal.,2018)mayimprovetheassessmentoftheC8dermatome.

Second, single trial analysis improved the relationship between CHEPs,specificallyN2P2amplitudeandP2latency,andage.Numer- ous studies have highlighted this relationship previously, generally confirmingthatnociceptive evokedpotentialsaresmallerandlonger with advanced age (Creac’H et al., 2015; Di Stefano et al., 2017; Granovsky et al.,2016; Lagerburg etal., 2015; Rosner etal., 2018; Truinietal.,2005).Thisisthoughttoprimarilyreflect aprogressive lossofnociceptorsintheperiphery(Ceballosetal.,1999;Ochoaand Mair,1969;O’SullivanandSwallow,1968;Yezierski,2012)andare- ductioninconductionvelocityofthespinothalamictract(Kakigiand Shibasaki, 1991), which are both paralleled by changes observed for other measures of pain (e.g.,thresholds) (Chakour et al., 1996; Gagliese,2009;GibsonandFarrell,2004; GibsonandHelme,2001).

Acrossstudies,however,thedetailsoftherelationshipbetweenageand

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Fig.7. Simulationstudyﬁndings:asmallwaveformandlargewaveform(40%largeramplitude,20msearlierlatency)wereusedtocreate16datasets.Theratio oflargetosmalltrialsisgiveninthex-axisofpanelsA,B,andC(e.g.5:10equateto5largewaveformsand10smallwaveforms).Conventionalaveraging(CA) andsingletrialanalysis(STA)N2P2outcomesfromeachdatasetwerecomparedforeachdataset.A)N2latenciesacrosssimulateddatasets.B)P2latenciesacross simulateddatasets.C)N2P2amplitudesacrosssimulateddatasets.D)Individualtrialsfrom5:10simulateddataset,boldlineisconventional(across-trial)average.

E)Individualtrialsfrom10:5simulateddataset,boldlineisconventional(across-trial)average.F)Across-trialaveragesfromallsimulationdatasets,boldlinesare theallsmallwaveformandalllargewaveformdatasets.

nociceptive evokedpotentialsareless consistent.For example,studies have reported an association for amplitudeonly (Frasson et al., 2020;Truinietal.,2005),whileothersobservedlocationdependence (Creac’Hetal.,2015;Granovskyetal.,2016;Rosneretal.,2018).Our observationssuggesttheoptimalapproachtodetectgenuineagerelated changesinnociceptive evokedpotentials, unrelatedtoslightaﬀerent desynchronizationandincreasedlatencyjitter,iswithsingletrialanal- ysis.

Finally, ratings were more strongly correlatedwith CHEPs from single trial analysis compared toconventional averaging. As a gen- eralruleofthumb,N2P2amplitudesarelargerandlatenciesshorter whenstimulationsare,onaverage,moreintense(Jutzeleretal.,2016; Krameretal.,2013,2012a;Lindeetal.,2020),albeitwithin-subjects (HuandIannetti,2019).Whileexceptionsarecommonplaceandhave aneuroanatomicalbasis(Krameretal.,2016),therelationshipbetween CHEPsandratingsreflectsanumberofimportantprocesses,including attention,arousal,saliency,andstimulusnovelty(Iannettietal.,2008; LePeraetal., 2002;Madsenetal., 2014;Ronga etal., 2013).Such endogenouscontributionsofevokedpotentialsarethoughttobemore susceptibletolatency jitter(Kutas etal., 1977; Legrainetal., 2002; SiedenbergandTreede,1996).Tothisend,methodsthatbettercap- ture arelationship withpain,aswas thecase for singletrialanalysis,arehighlydesirable.Theinherent valueofsingletrialanalysisis themoreaccurateportrayalofnociceptiveevokedpotentials.Similar topreviousstudies,weobservedsignificantlylargerN2P2amplitudes (Huetal.,2011)andlongerN2latencies(Warbricketal.,2009)follow- ingsingletrialanalysiscomparedtoconventionalaveraging.Increased amplitudesareattributabletophasecancelationresultingfromtrialto trialwaveformvariability(i.e.,latencyjitter),whichleadsto“flatten- ing” ofthegrandaveragewaveformthatservesthebasistointerpret conventionallyaveragedCHEPs(Huetal.,2011).IncreasedN2laten- ciesareattributedtodistortionsinwaveformmorphologyintroduced

byconventionalaveraging(Mayhewetal.,2006;Warbricketal.,2009).

Morespeciﬁcally,largeamplitude,individualwaveforms(i.e.,outliers), whichtendalsotobe shorter(Huetal.,2011;Iannettietal.,2005),

“pull” thegrandaverageleft,biasinginterpretationofconventionally averagedlatency.Wedemonstratedthisphenomenonwithsimulated data.Whenalownumberoflargeamplitudetrials,withearlierlatency, wereincludedinadatasetwithpredominatelysmalltrials,conventional (across-trial)averagedN2latenciesprogressivelyshiftedlefttoagreater extentthansingletrialaveragedlatencies(Fig.7A).Thisdifferencein N2latencycanbeexplainedbythedistortionorwaveformmorphology inconventionalaveraging,whichputsgreaterweightonlargerampli- tude waveforms.Incontrast,singletrialaveragingaccountsforthese differences in waveformmorphologyinthedeterminationindividual trialN2latencies,whicharesubsequentlyaveraged.Wealsoprovided anexampleofthisdifferencebetweenanalysismethodsasindividual trialsareaddedstepwise(Fig.8),againdemonstratedtheeffectoflarge amplitudetrialsinfluencingacrosstrialaveraging.Inaddition,thenon- linearincreaseinN2P2withconventionalaveragingprovidedfurther evidenceofdistortionsofwaveformmorphology,whichhavepreviously reported(Mayhewetal.,2006;Warbricketal.,2009).

C6CHEPsappeartohavebeenmoreaﬀectedbysingletrialaverag- ingcomparedtoC8(i.e.,largerincreaseinC6N2latency),whichmay relatetophysicaldiﬀerencesintestingsites.Onthedorsumofthehand, wherestimulationyieldsmorerobustCHEPscomparedtostimulationof thepalmersurface(Haefelietal.,2013b),theC8siteissmallerthanC6.

Thesizeofthestimulationsiteisamajorissueforcontactheatbecause thethermodeiscomparativelylargeandneedstobesubtlyshiftedafter eachstimulationwhilstremaininginthetargetdermatome.Thisisim- portantforclinicalapplications,whichaimtoassesssegmentalpatholo- gies(Haefelietal.,2013a;Krameretal.,2012a).InC6,shiftingthether- modeismorelikelytoactivatenovelreceptors,inturnproducingmore

“large” amplituderesponsesinC6(i.e.,outliers),whichultimatelyleads

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Fig.8. Conventional(arithmetic)averageandsingletrialaveragedN2latenciescomparedduringstepwiseadditionofindividualtrials.N2latencydenotedinred.

Itcanbeobservedthatwithsubsequenttrials,conventionalaveragingispulledfurtherleft,duetodiﬀerencesintrialamplitude.

toalargerdiscrepancybetweensingletrialandconventionallyaveraged CHEPs.

4.1. Limitations

Amajorstrengthofouranalysisisoursamplesize,whichislargein comparisontopreviousstudiesapplyingsingletrialanalysis(Huetal., 2010;Krameretal.,2016,2013;Mayhewetal.,2006;Warbricketal., 2009).Moreover,bothmenandwomenofvaryingageswereincluded.

Nevertheless,therearelimitations.Singletrialanalysisinvolvesaseries ofsignalprocessingsteps,whichultimatelyimprovesignalclarityofthe N2P2waveformbeforeextractingkeyamplitudeandlatencyoutcomes.

Giventhereareaseriesofstepsinvolved,thesemay,inpart,alsoen- hancethesignalclarityofN2P2waveforms.Forexample,convention-

allyaveragedCHEPswerenotwaveletfiltered.Assuch,ourfindings arelimitedtoacomparisonoftwooverallmethodsofdataanalysis.An- otherimportantpointisthatourfindingsarelimitedtoacomparisonof twoseparatemethodsofCHEPsanalysis,withnoobjective‘goldstandard’.Whileweprovideevidenceandsupportfortheuseofsingletrial analysis,thereremainsnotrue‘goldstandard’approachforCHEPsanal- ysis.Ourfindingsarealsolimitedtohealthysubjects.Furtherresearch isneededtodetermineifsingletrialanalysisimprovesunderstanding ofpathologyinpatientpopulations.

5. Conclusion

CHEPsprovideamethodtoreliablyandsafelyassesssmalldiam- eter nociceptiveaﬀerentsofthespinothalamicpathway thataretyp-

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icallyinvolvedin peripheralsensitization andchronicpaindevelop- ment.Whenoptimalstimulationanddataprocessing parametersare employed,CHEPoutcomesdemonstrateclear,robustage-andlocation- dependentchanges.Ourreportedimprovedassociationstoagingand ratingofperceivedintensitywhenusingsingletrialaveragingsuggest abetterrepresentationoftheunderlyingphysiologyofthenociceptive systemcomparedtotraditionalacross-trialaveraging. Whileconven- tional(across-trial)averagingoﬀersconvenienceandeaseofuse,itmay bemoresusceptibletowaveformdistortionandlatencyshiftswhenam- plitudevariationispresentamongindividualtrialsareincludedinanal- ysis.Werecommendtheuseofsingle-trialaveraging,withfreelyavail- ablesoftware(Huetal.,2011,2010),toassessthenociceptivesystem usingCHEPsinbothclinicalandresearchsettings.

CRediTauthorshipcontributionstatement

CatherineR.Jutzeler:Conceptualization,Methodology,Investiga- tion,Datacuration, Writing- review&editing, Fundingacquisition, Validation,Visualization.LukasD.Linde:Conceptualization,Method- ology,Formalanalysis,Writing-originaldraft,Writing-review&editing,Software,Visualization.Jan Rosner:Investigation,Writing -review&editing.MichèleHubli:Investigation,Writing-review&editing.ArminCurt:Supervision,Writing -review&editing. JohnL.K.

Kramer:Conceptualization,Writing-review&editing,Supervision,Re- sources,Projectadministration.

Acknowledgements

TheauthorswouldliketothankJanoshRinertforthesupportindata collection.CRJissupportedbytheSwissNationalScienceFoundation (AmbizioneGrant,PZ00P3_18610).Thisworkwasfurthersupportedby theSwissSpinalCordInjuryCohortStudyNestedProjectGrant(J.R.and C.R.J.,2016-N-005).JRissupportedbytheClinicalResearchPriority ProgramoftheUniversityofZurich(CRPPPain)andthroughfunding fromtheHartmannMuellerFoundation(grantnumber1997).Thefun- dershadnoroleinstudydesign,datacollectionandanalysis,decision topublish,orpreparationofthemanuscript.

Dataandcodeavailabilitystatement

Fullyanonymizeddatawillbesharedattherequestfromanyquali- ﬁedinvestigator(pleasecontacttheCorrespondingAuthor).Thecodeto runtheanalysisaswellascreatetheﬁgurescanbefoundonourGithub repository(https://github.com/jutzca/CHEPs_STA).

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