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Citation:Reiser,J.;Kreuzer,M.;Werner,J.;Saller,A.M.;Fischer,J.;Senf,S.;Deffner,P.;Abendschön,N.;Groll,T.;Grott,A.;etal.Nociception-InducedChangesinElectroencephalographicActivityandFOSProteinExpressioninPigletsUndergoingCastrationunderIsoﬂuraneAnaesthesia.Animals2022,12,2309.https://doi.org/10.3390/ani12182309AcademicEditors:PeterWhite,DominiqueVanderSaagandGabrielleC.MuskReceived:9August2022Accepted:31August2022Published:6September2022Publisher’sNote:MDPIstaysneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalafﬁl-iations. Copyright:©2022bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution(CCBY)license(https://creativecommons.org/licenses/by/4.0/). ArticleNociception-InducedChangesinElectroencephalographicActivityandFOSProteinExpressioninPigletsUndergoingCastrationunderIsoﬂuraneAnaesthesiaJudithReiser1,MatthiasKreuzer2,JuliaWerner1 ,AnnaM.Saller1 ,JohannesFischer1,SteffanieSenf3 ,PaulineDeffner3 ,NoraAbendschön3,TanjaGroll4 ,AndreaGrott5,ReginaMiller5,ShanaBergmann5,MichaelH.Erhard5 ,MathiasRitzmann3,SusanneZöls3,GerhardSchneider2 ,KatjaSteiger4,† andChristineBaumgartner1,*,†1CenterofPreclinicalResearch,TechnicalUniversityofMunich,81675Munich,Germany2DepartmentofAnesthesiologyandCriticalCare,SchoolofMedicine,TechnicalUniversityofMunich,81675Munich,Germany3ClinicforSwine,CenterforClinicalVeterinaryMedicine,LMUMunich,85764Oberschleissheim,Germany4InstituteofPathology,SchoolofMedicine,TechnicalUniversityofMunich,81675Munich,Germany5ChairofAnimalWelfare,Ethology,AnimalHygieneandHusbandry,LMUMunich,80539Munich,Germany*Correspondence:christine.baumgartner@tum.de†Theseauthorscontributedequallytothiswork.SimpleSummary:Efﬁcientanalgesiaduringsurgicalpigletcastrationisanimportantanimalwelfareissue.Thepresentstudyispartofalargerstudydesignedtoinvestigatetheefﬁcacyoffourlocalanaesthetics(bupivacaine,lidocaine,mepivacaineandprocaine)foranalgesiaduringcastration.Inconsciouspiglets,itisdifﬁculttodistinguishbetweenstress,e.g.,duetohandling,andpainduringcastration.Therefore,thisstudywasconductedunderlightisoﬂuraneanaesthesia.Thelevelofanaesthesiawasadjustedsuchthatmovementreactionstoaperipheralnociceptivestimulusstilloccurred.Wepresenttheresultsoftheinvestigationoftwopossibleparametersforthedetectionofnociception:thereactionpatternintheelectroencephalogram(EEG)andFOSproteinexpressioninthespinaldorsalhorn.Intheelectroencephalogram,abiphasicreactionpatterninresponsetonoxiousstimulationwasdetectedthatwasattenuatedoralteredbytheapplicationoflocalanaes-thetics.FOSexpression,whichwasexaminedpostmortem,wasdecreasedaftertheadministrationoflocalanaesthetics,exceptforbupivacaine.Basedontheseresults,localanaesthesiadecreasesnociceptivetransmissionduringpigletcastrationinthisexperimentalsetup.Whencombinedwiththecorrespondinghaemodynamicparametersandtheevaluationofdefensivemovementspresentedelsewhere,anoverallunderstandingofthenociceptiveresponsetocastrationcanbegenerated.Abstract:TheobjectiveofthisstudywastoinvestigatetheelectroencephalographicreactionpatternandFOSproteinexpressioninmalepigletsundergoingsurgicalcastrationunderlightisoﬂuraneanaesthesiawithorwithoutlocalanaesthesia.Theexperimentwasconductedunderisoﬂuraneanaesthesiatoexcludetheeffectoftheaffectivecomponentsofpainonthemeasurements.Changesintheoscillatoryactivityofthecerebralcortexovera90speriodafternoxiousstimulationorsim-ulatedinterventionswereanalysed.FOSexpressionwasdeterminedpostmortembyperformingimmunohistochemistryinthedorsalhornofthespinalcord.Theanalysisoftheresponsetoaninterdigitalpinchrevealedabiphasicreactionpatternintheelectroencephalogram(EEG)thatsim-ilarlywasobservedforthesurgicalstimuliduringthecastrationprocedureinthegroupwithoutanalgesia.ThisEEGresponsewasattenuatedoralteredbytheapplicationoflocalanaesthetics.ImmunohistochemicalstainingforFOSindicatedalowerexpressioninthehandlingandinthreelocalanaestheticgroupsthanintheanimalscastratedwithoutpainrelief.TheﬁndingsindicatethatEEGandFOSexpressionmayserveasindicatorsfornociceptioninpigletsunderlightisoﬂuraneanaesthesia.Aloweractivationofnociceptivepathwaysoccursduringcastrationaftertheappli-cationoflocalanaesthetics.However,EEGandFOSanalysesshouldbecombinedwithadditionalparameterstoassessnociception,e.g.,haemodynamicmonitoring. Animals2022,12,2309.https://doi.org/10.3390/ani12182309https://www.mdpi.com/journal/animals Animals2022,12,2309 2of17 Keywords:EEG;FOS;piglet;electroencephalography;castration;localanaesthesia;nociception;lidocaine;procaine;mepivacaine;bupivacaine;spectrogram;animal 1.IntroductionSurgicalcastrationofmalepigletsoflessthaneightdaysofagewithoutanaesthesiahasbeenacommonpracticefordecadesinseveralcountriestopreventboar-taintedmeatandaggressivebehaviourofintactmalepigs,aswellastoobtaintherequestedmeatquality.However,surgicalcastrationwithoutpainreliefisapainfulintervention[1–3].Signiﬁcanteffortshavebeendevotedtodevelopingmethodsthatprovideeffectiveanalgesiaforthesurgicalcastrationofpigletsandtechniquesfortheexaminationofpainandnociceptionduringthisprocedure.Painisacomplexmultidimensionalexperienceinvolvingsensoryandaffective(emo-tional)componentsthatisonlyexperiencedbyconsciousanimalsandisasubjectiveemotion[4,5].Thepresentstudyaimedtomeasurenociception,thesensorycomponentofpain,andtoassesstheefﬁcacyoflocalanaestheticsduringthecastrationofmalepiglets.Anaesthesiawithisoﬂuranewasadministeredtoexcludetheeffectsofaffectivecompo-nentsonthemeasurements.Thus,theeffectsoffearandstressonmodulatingthepainexperienceduetotheconsciousexperienceofhandling,ﬁxationornoiseswereeliminated.Asisoﬂuranehasnoornegligibleanalgesicproperties,anociceptiveresponsewasstillpresent.Weaimedtoestablishadepthofanaesthesiaatwhichthenociceptivewithdrawalreﬂexwasstillpresent.EEGandFOSexpressionwereevaluatedattwodifferentlocationsinthenociceptivepathway:FOSexpressionwasmeasuredinthedorsalhornofthespinalcord,whichistheﬁrstrelaystationinnociceptivetransmissionofperipheralstimuli,andpain-inducedchangesincortical(electrical)activitywererecordedusinganEEG,representingtheﬁnalstageofnociceptivesignalling—thebrain.EEG,adirectmeasureofbrainelectricalactivity,enablestheimmediateassessmentofpossiblepain-inducedchanges.However,theappropriateidentiﬁcationofEEGchangescausedbynoxiousstimulationisdifﬁcultduetotheheterogeneityofpossibleEEGre-sponses[6,7].NumerousstudieshaveevaluatedchangesintheEEGsofanimalscausedbynoxiousstimulationundergeneralanaesthesia[8–16].Inpiglets,EEGmeasurementshavealsobeenconductedtoinvestigatethecorticalresponsestonoxiousstimulation.TheresultsdescribeEEGchangesfollowingcastration[17,18],exposuretoothersurgicalstimuli[19–21]andinnocuousnoxiousstimuli[22].Somestudiesinanimalshaverevealedreactions,whileothersfailedtoidentifychangesinEEGparametersfollowingnoxioussurgicalstimulationundergeneralanaesthesia.AllofthesestudiesusedprocessedEEGparameters,suchastotalpowerorspectraledgefrequencies,derivedfromtheEEGpowerspectrumtodescribetheirﬁndings.However,themereuseoftheseprocessedparameterscomplicatestheabilitytocompareﬁndingsfromdifferentstudies.Therefore,thegeneralspectralEEGresponsetonoxiousstimulationispresentedinadetailedmannerasdensityspectralarrays,anapproachthatdoesnotreduceinformationduetotheuseofprocessedEEGparameters.Peripheralnoxiousstimulationevokestherapidexpressionoftheimmediateearlygenec-FOSanditsproteinproductFOSinthenucleiofpostsynapticneuronsinthespinaldorsalhorn[23,24].Theexpressionofc-FOSandFOSinneuronsofthespinalcordisanestablishedmarkertoquantifyneuralactivityinresponsetonoxiousstimulationpostmortem[25,26].Itwasappliedinpigsaswell,andsinglestudieshaveevaluatedFOSexpressiontoinvestigatepainornociception,respectively,followingpigletcastration[27–31].Thegroupwithoutfurtheranalgesictreatment(NaCl)wascomparedwiththeanimalsthatreceivedlocalanaesthesiawitheither0.5%bupivacaine,2%lidocaine,2%mepivacaineor4%procaine,aswellaswiththeanimalsthatunderwentonlysimulatedinterventionstocorrelatetheobservedchangesinEEGpatternsandFOSexpressionwithnociception. Animals2022,12,2309 3of17 Inthisstudy,electroencephalographicreactionsinthecerebralcortexandchangesinFOSproteinexpressioninthespinaldorsalhornofsurgicallycastratedmalepigletsunderlightisofluraneanaesthesiaaredescribed.Thesemeasurementswereevaluatedasadditionalparametersofnociceptioninamultiparametricstudy.Thecorrespondingresultsforhaemo-dynamicandneurohumoralmeasurementsandtheevaluationoflimbmovements,whichwereinvestigatedinthesamepiglets,arepresentedinastudybySalleretal.[3].2.MaterialsandMethodsThestudywasperformedincompliancewiththeEUDirective2010/63/EUforanimalexperimentsandtheGermanAnimalWelfareAct[32].AllprocedureswereapprovedbytheEthicsCommitteeforAnimalExperimentsoftheGovernmentofUpperBavaria,Munich,Germany(ReferenceNumberROB-55.2-2532.Vet_02-19-11).2.1.AnimalsForty-ninehealthymalehybridGermanLandrace/GermanLargeWhite Pietrainpigletsfrom15littersagedthreetosevendayswereincludedinthisstudy.PregnantsowsweresourcedfromacommercialpigletproducerandtransferredtotheanimalhusbandryunitoftheClinicforSwine(CenterforClinicalVeterinaryMedicine,LMUMunich,Oberschleissheim,Germany).SowsandpigletswerehousedaccordingtotheGermanOrderfortheKeepingofProductiveAnimalsandtheEUDirective2010/63/EUforanimalexperiments.Usingacomputer-generatedrandomisedgroupassignment,animalsweredistributedintosixtreatmentgroups:fourgroupsreceivedeither4%procainehydrochloride(1),2%lidocainehydrochloride(2),0.5%bupivacainehydrochloride(3)or2%mepivacainehydrochloride(4)asalocalanaesthetic,onegroupreceivedaninjectionofsalinewithoutfurtheranalgesia(5)andonehandlinggroupunderwentonlysimulatedinterventions(6).Thestudywasdesignedasarandomised,double-blindstudy.2.2.ExperimentalProtocolAnaesthesiawasinducedwithaninitialconcentrationof5%isoﬂurane(IsoﬂuraneBaxtervet.,BaxterDeutschlandGmbH,Unterschleissheim,Germany)in3L/minoxygenviaafacemask.Pigletsbreathedspontaneouslyduringtheentiretrial.Whenthemusclesrelaxedandtherightingreﬂexwasnolongerelicited(noreactionwhentheanimalwasplacedinthesupineposition),theconcentrationofinspiratoryisoﬂuranewasreduced.Aftertheinductionofanaesthesia,ﬁve27-gaugedisposablestainlesssteelneedleelectrodeswereplacedsubcutaneouslyontheheadofthepigletasfollows:recordingelectrodeswereplacedsupraorbitallyintheareaofthefrontalboneonbothsidesofthehead,tworeferenceelectrodeswereplacedsidebysideinthemidlineoftheheadatthehighestpointoftheskull,andthegroundelectrodewasplacedintheneck.CorticalsignalswererecordedcontinuouslythroughouttheexperimentwithacommercialEEGmonitor(Narcotrend©,MTMonitortechnikGmbH&Co.,KG,BadBramstedt,Germany).EEGdatawererecordedfromtwobipolarchannelswithasamplingrateof128Hz.AfterplacingtheEEGelectrodes,thepigletwasturnedtothesupinepositiontoinsertcathetersintothecarotidarteryandthejugularveintomonitorbloodpressureandheartrateandwithdrawbloodsamples.Duringallnoxiousinterventions,theoccurrenceoflimbmovementswasalsorecorded(adetaileddescriptionoftheprocedureandresultshasbeenpublishedbySalleretal.[3]).Followingthemeasurementpreparations,inspiratoryisoﬂuranewasfurtherreducedto1.5%.Theappropriateconcentrationofisoﬂurane(presenceofnociceptivewithdrawalreﬂex)wasevaluatedineverypigletindividuallybyapplyingaPeanclamptopinchtheinterdigitalskinfoldofthehindlimb.Theclampwasclosedmaximallytotheﬁrstratchetforamaximumtimeof5soruntilmovementwaselicited,whicheverperiodwasshortest.Ananaestheticlevelallowingforaslightmovementreactionofthestimulatedlimb,provokingneitherinappropriatelyintensemovementreactionsnorawakeningthe Animals2022,12,2309 4of17 piglet,wasdeterminedtobeappropriate.Ifnomovementreactionoccurred,theinspiredisoﬂuraneconcentrationwasreducedin0.2%stepsandsubsequentlyadjusteduntilaslightlimbmovementoccurred.Incontrast,prolongedpaddlingand/ormovementoftheforelimbs,backorheadwereassessedasbeingtooexcessivefortheexperimentalsetup,andtheinspiredisoﬂuraneconcentrationwasincreasedinstepsof0.2%andequili-bratedfor3min.Thisapproachresultedinameanend-tidalisoﬂuraneconcentrationof1.69 0.3%forcastration.Aftertheadequatehypnoticstatewasreached,aninjectionofeitherlocalanaestheticsorsalinewasperformed,exceptinthehandlinggroup,whereinjectionsandsurgicalinterventionswereonlysimulated.ThetimelinefortheoverallexperimentispresentedbySalleretal.[3].Allpiglets,exceptforthehandlinggroup,receiveda0.5mLintratesticularanda0.5mLsubscrotalinjectionpertestis.Theappliedlocalanaestheticswere0.5%bupivacainehydrochloride(Bupivacain0.5%,JENAPHARM,MibeGmbHArzneimittel),2%lidocainehydrochloride(Xylocitin®2%,MibeGmbHArzneimittel,Brehna,Germany),2%mepiva-cainehydrochloride(Mepidor®20mg/mLsolutionforinjectioninhorses,RichterPharmaAG,Wels,Austria)and4%procainehydrochloride(4%procainehydrochloride-VMD,V.M.D.sa,Arendonk,Belgium).ThepHvaluesofthefourlocalanaestheticsrangedfrom3to6.5(pHvaluesaccordingtothemanufacturer:4%procainehydrochloride3–4.5,0.5%bupivacaine4–6,Mepidor®~5.4,and2%Xylocitin®5.5–6.5).Twocontrolgroupswereincludedinthestudy:onegroupthatreceivedasalineinjection(0.9%NaCl,castrationwithoutpainrelief)andtheshamgroupthatunderwentonlyasimulatedinjectionandcastration(handling).Anautomaticself-ﬁlling1mLsyringe(HSWECO-MATIC®,Henke-Sass,WolfGmbH,Tuttlingen,Germany)witha25Gcannula(0.5 16mm,B.BraunTravaCareGmbH,Hallbergmoos,Germany)wasusedfortheinjection.Thetestiswasﬁxedwiththethumbandindexﬁngerfortheintratesticularinjection.Subsequently,asmallskinfoldwasformedoverthetestis,whilethecannulawasretracteduntilthetipwaslocatedsubcutaneously,andthesubscrotalinjectionwasperformed.Thehandlinggroupwasmanipulatedandﬁxedinthesamemannerastheothergroups.Theautomaticsyringewaspushedonlyslightlyagainstthetesteswiththeneedlecappedtosimulatetheinjectionsinthisgroup.Afteranexposuretimeof20min,surgicalcastrationwasinitiated(exceptinthehandlinggroup).Inthecaseofcastration,theskinwasopenedwithascalpelbycreatingtwosagittalincisionsdowntotheviscerallayerofthevaginaltunic,andthetesteswereexposed.Afterastabilisationperiodoftwominutes,thetestesweregrasped,andthespermaticcordsweresuccessivelyseveredwithanemasculator.Thebluntbackofascalpelhandlewasmovedoverthescrotumtoimitatetheskinincisionintheanimalsofthehandlinggroup.Anaesthesiawasmaintainedforanother90minbeforeeachpigletwaseuthanisedwithanintravenousoverdoseofpentobarbital(Euthadorm500mg/mL,CPPharma,Burgdorf,Germany).FortheevaluationofEEGreactions,theEEGrawtraceswerevisuallyinspectedforartefactsorEEGburstsuppression(BSupp)patternsofﬂine.BecauseBSuppEEGcharacteristicsstronglydeviatefromtheEEGpatternspresentundergeneralanaesthesiawithoutBSupp,thedecisionwasmadetoexcludethesesegmentsfromfurtheranalysis.Allanimalsreceivedtheentiresetofnoxiousstimuliexceptforthehandlinggroup,inwhichonlyinterdigitalpinchesandsimulatedinterventionsweretested.Inthepigletsthatunderwentmorethanoneinterdigitalpinchfortheadjustmentofanaestheticdepth,thepinchthatwasincludedintheanalysiswastheoneatwhichtheanaestheticdepthwasevaluatedtobeappropriate.Eachbilateralinterventioninjection,skinincisionandcuttingofthespermaticcordwasperformedwithinafewsecondsonbothtestes.Theinjectionrequiredapproximately9stocompletepertestis,andskinincisionsanddissectionsofthespermaticcordrequired Animals2022,12,2309 5of17 approximately6seachtocomplete.TheEEGwasanalysedoveratimespanof90sfollowingthebilaterallyperformedstimulitofullyassesstheresponsetothenociceptivestimulus.Immediatelyaftereuthanasia,lumbarandsacralsegmentsofthespinalcordweredissectedfromthesurroundingtissueandremovedforFOSmeasurements.Usingthevertebraeaslandmarks,thespinalcordwascutintothreetransverselumbarsegments(L1,L2,andL3)andonesacral(S1-S3)spinalcordsegment,andﬁxedwith38%(w/w)neutral-bufferedformaldehydeforatleast48handembeddedinparafﬁn(LeicaASP300S,Leica,Wetzlar,Germany).Consecutivesections(2 m)wereobtainedfromthecranialsectionofeachparafﬁnblockandstainedwithhaematoxylin–eosin.FOSimmunohistochemistry(anti-c-FOSantibody,ab209794,Abcam,Cambridge,UK,dilutedinantibodydiluent1:100)wasperformedusingaLeicaBondRXmsystem.Brieﬂy,afterdeparafﬁnisation,pretreatmentwasperformedwithEpitopeRetrieval1(correspondingtocitratebufferpH6)for30min.Forprimaryantibodybindingdetectionandvisualisation,apolymerreﬁnedetectionkitwasusedwithoutapostprimaryantibodyandwith3,3’-diaminobenzidine(DAB)asthechromogen.AllslideswerescannedwithaLeicaAT2scanningsystem.Averticallinewasdrawnfromthecentralcanaltothelateralborderofthegreymattertostandardisetheareaforanalysis.Thespinaldorsalhornwasdeﬁnedasthegreymatterdorsaltothelinethatwasdrawn(Figure1B).ThestainingintensityandfrequencyofFOS-positiveneuronsinthespinaldorsalhornwereevaluatedsemiquantitatively(Figure1)byaGermanboard-certiﬁedpathologistwhowasblindedtothegroupsusingAperioImagescopeSoftwareversion12.4.0.7081. Figure1.SemiquantitativeEvaluationoftheFOSStainingIntensity.(A)ScorefortheFOSstainingintensityandfrequency.(B)Areaevaluatedinthespinalcord.(C)Examplesofdifferentscores.Theanalysismethodwasﬁrstestablishedinapreliminarystudy(datanotshown)todevelopanddeﬁneascoringapproachforformalin-ﬁxed,parafﬁn-embeddedandstainedtissuesections.Thestainingintensity(none,slight,moderate,orstrong)andthepercentageofpositiveneuronalnucleiinbothdorsalhornsofthespinalcordswerecalculated.Asemiquantitativescoreincludingthesetwoparameters(Figure1A)wasapplied.Fourdifferentsegments(L1,L2,L3,andS1-3)ineachanimalwereevaluated.Theresultsofthisevaluationclearlyshowedthehighestscoresintheanimalscastratedwithoutpainrelief.Thus,itwasdecidedtoproceedwiththismethodologicalapproach. Animals2022,12,2309 6of17 2.3.StatisticalAnalysis2.3.1.EEGThepowerspectraldensityoftheselectedEEGepisodeswascalculatedwiththeMATLABpwelchfunctionandafrequencyresolutionof0.25Hzforthepooleddatafromtheinterdigitalpinchstimulusand1Hzforthedatarecordedinresponsetootherstimuli.Becauseofthelargersamplesizeinthepooledgroupforthepinchstimulus,wedecidedtouseahigherfrequencyresolutiontobetterdescribetheresponse.Densityspectralarrayswerealsoconstructed,andtheresultsarepresentedasheatmapstodepictthetemporalevolutionofthespectralEEGfeatures.TheDSAwasderivedfrom5-sEEGepisodesoverlappingby4s.TheresultsarepresentedaschangesintheEEGpowerrelativetoaprestimulusbaselinedeﬁnedastheepisode50–40sbeforethestimulus.Thepoststimulusvaluewasdividedbytheprestimulusvalue.Thus,avalue<1indicatesahighervaluebeforethestimulus,whereasavalue>1indicatesanincreaseafterstimulation,andavalueof1indicatesnochangeafterthestimulus.Thisapproachhelpstotrackstimulus-inducedEEGchangesandcorrectfordifferentEEGamplitudesbetweentheanimals.FortheDSAplots,asignedranktestwasperformedforeachpixelbyassessingthepoweroftherespectivefrequencyattheobservedtimeversusthepowerofthefrequencyatbaseline.Pixelsthateitherhadap<0.05(pooledresponsetotheinterdigitalpinch)orp<0.1(singleexperimentalgroup,allotherstimuli)areshown.Hence,onlysigniﬁcantchangesthatoccurredinclustersarediscussed.Theclusterapproachwasusedbecauseofthelowprobabilityoftheconcentrationofspuriousfalsepositivesinacluster.Therefore,thestrategywasusedtodeﬁneaclustersize[33],i.e.,eithera4s 2Hzclusterforthepooledreactiontotheinterdigitalpinchstimulusora4s 4Hzclusterforthereactiontootherstimuli.Theareaunderthereceiveroperatingcurve(AUC)wascalculatedwith10k-foldbootstrapped95%conﬁdenceintervalstotrackthechangesintheEEGbandpowerovertime.Ifthe95%conﬁdenceintervalsdidnotcrossthe0.5line,thisresultwasconsideredtobesigniﬁcantaslongasitwasobservedinatleastattwoneighbouringtimepoints.Similarapproacheshavebeenusedpreviously[34,35].MATLABR2017a(TheMathWorks,Inc.,Natick,MA,UnitedStates)wasusedforthestatisticalanalysis.FortheAUCcalculation,additionalfunctionsfromthemeasuresoftheeffectsizetoolboxwereused[36].Differentsettingswereusedfortheinterdigitalpinchstimulusandtheothernoxiousstimuli,asamuchlargernumberofsampleswasavailablefortheinterdigitalpinchbypoolingdatafromallanimals,independentofthetreatmentgroups.BeeswarmplotsweregeneratedwiththeMATLABplotSpreadfunction.2.3.2.FOSProteinFourtissuesectionsperanimalwereincludedintheanalysis:onesectioneachfromspinalcordsegmentsL1,L2,L3andS1-3.Themedianstainingintensityandfrequencyofthedark-brownDABprecipitateinthefourspinalcordsegmentswerecalculatedforeveryanimal,andaKruskal–WallistestwasappliedtoidentifysigniﬁcantdifferencesbetweentheNaClgroupandtheotherexperimentalgroups.Figure1showsthescoringschemeappliedfortheFOSanalysis.3.Results3.1.DemographicsFortheEEGanalysis,196datasets(fourperanimal)from49pigletsaged5.3 1.1daysandweighing2.14kg 0.45kgwereacquired.SomeanimalsexhibitedtimespanswithBSuppinmeasurementperiods;therefore,30datasetswerediscarded(27duetoBSupppresenceand3duetoartefacts),and166datasetswereincludedintheanalysis.Theinter-digitalpinchwasperformedunderisoﬂuraneanaesthesiaintheabsenceofanyanalgesic.Itwasappliedtoallanimalsusinganidenticaltechnique.Therefore,theanimalsfromallexperimentalgroupswerecombinedtoevaluatetheEEGchangesduetotheinterdigitalpinch(n=44datasets).Forthesereasons,varyingnumbersofdatasetswereanalysedforthesixexperimentalgroups(Table1). Animals2022,12,2309 7of17 Table1.Overview:NumberofDatasetsUsedintheEEGAnalysis. ExperimentalGroup NoxiousStimulus InterdigitalPinchInjectionIncisionCuttingoftheSpermaticCord NaCl(n=8animals) n=44datasets(pooleddata)n=7datasetsn=6datasetsn=6datasetsLidocaine(n=9animals) n=7datasetsn=8datasetsn=7datasetsBupivacaine(n=9animals) n=9datasetsn=9datasetsn=9datasetsMepivacaine(n=6animals) n=5datasetsn=5datasetsn=5datasetsProcaine(n=8animals) n=6datasetsn=5datasetsn=5datasetsHandling(n=9animals) n=7datasetsn=8datasetsn=8datasets Totaln=49animals Totaln=166datasets FortheFOSanalysis,tissuesectionsfrom42animalswereevaluated.Duetosamplingandtrimminginaccuracies,theindividualspinalcordsectionsfromsevenanimalscouldnotbeevaluated.Therefore,thosesevenanimalswereexcludedfromtheﬁnalanalysis.Theanalysedanimalswere5.5 1.0daysofageandweighed2.13kg 0.45kg(Table2).Table2.Overview:NumberofAnimalsfortheFOSAnalysis. FOSproteinAnalysisNaClLidocaineBupivacaineMepivacaineProcaineHandling (n=42animals)n=7n=7n=8n=7n=6n=7 3.2.InterdigitalPinchForthepooleddata(n=44),asigniﬁcantincreaseinfrequenciesof~10–14Hzwasobservedimmediatelyafterthestimuluswasapplied,alongwithasigniﬁcantdecreaseinthe~0.5–8Hzbandpower(deltaandthetarange,‘early’response).Thedecreaseinpowerlasteduntilapproximately35safterthebeginningofstimulation(t=0).Afterthisdecreaseinpower,anincreaseinpoweratfrequencies>10Hzwasobservedstartingafterapproximately35suntilapproximately90saftert=0(‘late‘response).Figure2presentstheDSAofthechangeinpowerrelativetothebaselineandhighlightstheearlyandlatecomponentsobservedfollowingtheinterdigitalpinch. Figure2.DSAofthePooledMedianResponse(n=44)toInterdigitalPinchStimulationover90s.Areaswithboldcoloursindicatethefrequencyandtimeregionswithsigniﬁcantdifferencesbetweenpre-andpoststimulusconditions.Thecolourbarindicatesthechangeinthepoststimuluspowercomparedwiththeprestimulusbaseline.Thestimulusstartedatt=0.Brieﬂy,theinterdigitalpinchleadstoan‘early‘reductioninpowerintheslowfrequenciesbelow10Hzanda‘late‘activationofhigherfrequenciesabove10Hzstartingapproximately35safterthestimulation.3.3.InjectionsThereactionstotheinjectionstimuliwerequiteheterogeneousamongthesubstances.FortheNaCl,bupivacaineandprocainegroups,asigniﬁcantreactionwasobservedasanactivationoffrequenciesof~10–15Hzatapproximately30–40safterthestimulus.Inparticular,intheprocainegroup,asigniﬁcantongoingdepressionof~10–15Hzoscillationsappearedstartingapproximately60safterthestimulus.Forthemepivacaineandprocainegroups,areductioninpowerinthelowerfrequenciesimmediatelyaftertheinjectionstimuluswasdetected.Lidocaineandbupivacaineproducedshort,signiﬁcantdecreasesatapproximately80s,atfrequenciesof~10–15Hz. Animals2022,12,2309 8of17 SupplementalFigureS1presentstheDSAsofthechangesinpowercomparedwithprestimulusconditionsforthe90speriodaftertheintratesticularandsubscrotalinjections(FigureS1).3.4.SkinIncisionAbiphasic,earlyandlateresponsetothescrotalskinincisionwasobservedintheNaClgroupwithan‘early’(~10s)decreaseof~5–15Hzpowerfollowedbya’late’activationof~20–30Hzpower,startingatapproximately25s.Forallothergroups,asignificant,stimulus-inducedchangewasnotobserved.Figure3presentstheDSAsofthechangeinpowerwhencomparedwiththeprestimulusconditionsforthe90speriodaftertheincision. Figure3.DSAsoftheChangesinEEGsaftertheScrotalSkinIncision.TheDSAspresentchangesinEEGpowerover90sfollowingtheincisionstimulusinthefollowingexperimentalgroups:NaCl(A),lidocaine(B),bupivacaine(C),mepivacaine(D),procaine(E),andnoincisionandhandlingonly(F).Theincisionstartedatt=0.AreasshowninboldcoloursindicatefrequencyandtimeregionswithasigniﬁcantdifferencefromtheprestimulusEEG.(A)NaClcausedan‘early‘and‘late‘responsetotheincisionstimulus.Theearlyresponsewasanattenuationinthe5–15Hzrangewithintheﬁrst20safterstimulation,andthelateresponsewasanactivationinthe20–30Hzpowerrangeatapproximately30–40safterthestimulation.(B–F)SigniﬁcantchangesintheEEGpowerwerenotobservedinthelocalanaestheticandhandlinggroupsfollowingthestimulus,exceptforahigh-frequencyactivationatapproximately30Hzandapproximately60safterstimulationinthebupivacainegroup(C). Animals2022,12,2309 9of17 3.5.CuttingoftheSpermaticCordAnalogoustotheincisionandtheinterdigitalpinch,abiphasicresponsetospermaticcorddissectionwasdetectedintheNaClgroup,withanearly(~10s)decreasein~5–15Hzpowerfollowedbyalateactivationof~20–30Hzpowerstartingatapproximately20s.Thisbiphasicresponsewasnotobservedinanyoftheothergroups.Forthelidocainegroup,anincreasein~10Hzpowerwasobservedapproximately40safternoxiousstimulation.Forallothergroups,nospeciﬁcreactiontothestimuluswasdetected.Figure4presentstheDSAofthechangeinpowerforthe90speriodafterspermaticcorddissection,comparedwiththeprestimulusconditions. Figure4.DSAsoftheEEGChangesafterSpermaticCordDissection.TheDSAspresentchangesinEEGpowerover90sfollowingspermaticcorddissectioninthefollowingexperimentalgroups:NaCl(A),lidocaine(B),bupivacaine(C),mepivacaine(D),procaine(E),andnoincisionorhandlingonly(F).Stimulusstartedatt=0.(A)IntheNaClgroup,an‘early‘and‘late‘responsetothedissectionstimuluswasobserved.Theearlyresponsewasanattenuationinthe~5–15Hzrangewithintheﬁrst15safterstimulation,andthelateresponsewasanactivationof~20–30Hzatapproximately20and30–40safterstimulation.(B–F)NosigniﬁcantchangesintheEEGpowerwereobservedinthelocalanaestheticandhandlinggroupsfollowingthestimulus,exceptforanactivationof~10Hzpoweratapproximately40safterstimulationinthelidocainegroup(B)andalatedecreasein~15Hzpoweratapproximately80sinthebupivacainegroup(C). Animals2022,12,2309 10of17 3.6.EvaluationoftheEarlyandLateResponsesTheNaClgroupshowedasimilarreactionpatternfollowingskinincisionandcuttingofthespermaticcord,namely,anearlydecreaseinlow-frequencywavesfollowedbyanincreaseinhigh-frequencywaves(lateresponse).ThedetectionperiodsofalllocalanaestheticgroupsandthehandlinggroupwerecomparedwiththoseoftheNaClcontrol.Figure5presentsbeeswarmplotsofthestatisticalanalysesoftheearly(5–10safterstimulus)decreaseinthefrequencybandsof5–15Hzandthelate(30–40safterstimulus)increaseinthefrequencybandsof20–30Hz.Thereactionsofthedifferentsubstancegroupsandthehandlinggrouptoskinincisions(Figure5A,B)andspermaticcorddissections(Figure5C,D)werecomparedwiththeNaClgroup. Figure5.Comparisonof‘early‘and‘late‘reactionsfollowingskinincision(A,B)andspermaticcorddissection(C,D),comparedwiththeNaClgroup.(A)Forskinincisions,asigniﬁcant(*)orpossiblyrelevant(AUC>0.7)effectwasobservedfortheearlyresponseinallgroups,exceptfortheprocainegroup.The5–15HzpowerdecreasedbyasmalleramountasareactiontothestimuluswhencomparedwiththechangeintheNaClgroup.(B)Regardingthelateresponse,allgroupsshowedaweakeractivationof20–30HzpowerasaresultofthestimulationcomparedwiththatoftheNaClgroup.(C)Asanearlyreactionfollowingspermaticcorddissection,allgroupsexhibitedaweaker5–15HzresponsethantheNaClgroup.(D)Signiﬁcantdifferencesinthelate20–30Hzpowerresponseswerenotobservedafterspermaticcorddissection.3.7.FOSProteinSigniﬁcantlylowerexpressionoftheFOSproteinwasobservedintheanalysedlumbar(L1,L2,L3)andsacral(S1-3)segmentsofthespinaldorsalhorninthehandlinggroupandinalllocalanaestheticgroups,exceptthebupivacainegroup,whencomparedwiththeNaClgroup.Allanimals,includingthehandlinggroup,displayedatleastslightstainingin<25%oftheneuronsthatresultedinaminimumscoreofone.Figure6displaysthecorrespondingscatterplotaswellasimagesoftheimmunohistochemicalstaining.Additionally,FOSproteinexpressionwasevaluatedonlyinanimalsthatshowednoBSupppatternsthroughoutthemeasuredEEGepochs(FigureS2). Animals2022,12,2309 11of17 Figure6.ScatterplotoftheFOSstainingscore(A)andrepresentativeimagesofFOSproteinexpression(B–E).(A)Signiﬁcantly(AUC95%CIexcept0.5)lessFOSproteinexpressionwasobservedinthehandlinggroupandinalllocalanaesthesiagroups,exceptforthebupivacainegroup,thanintheNaClgroup.(*)Asigniﬁcanteffect(p<0.05).(B–E)RepresentativeimagesofFOSproteinexpressioninthedorsalhornofthespinalcordinanimalsfromtheNaCl(B),lidocaine(C),bupivacaine(D),andhandling(E)groups(barsindicate30 m).4.DiscussionTheinitialanalysisofthechangesinEEGpatternsfollowingtheinterdigitalskinfoldpinch,whichinducedanociceptivewithdrawalreﬂex,showedastrongbiphasicresponse.Asthisstimuluswasappliedtoallanimalsinanidenticalmannerwithoutanalgesia,thedatawerepooled.Thisapproachyieldedalargenumberofsamples.ThebiphasicEEGresponseconsistedofan‘early’anda‘late’component.SimilarEEGreactionswererecordedfollowingthescrotalskinincisionanddissectionofthespermaticcordwithoutlocalanaesthetics.Theapplicationoflocalanaestheticsattenuatedcorticalreactions.Additionally,FOSproteinexpressioninthedorsalhornofthespinalcordwasreducedbymostlocalanaesthetics.Usinganinterdigitalpinch,whichwasreportedtobeastrongnoxiousstimulusinnewbornsandjuvenilepigs[37,38],theadequateanaestheticlevelforeachanimalwasdeterminedindividually.Thepresenceofanociceptivewithdrawalreﬂexinducedbyinterdigitalpinchingisawell-acceptedindicatorofnociceptionduringanaesthesiainvariousspeciesandinpigs[39–41].BSupp,apatternofwaxingandwaningamplitudes,mayreﬂectastateofexces-sivelydeepanaesthesia,butitmayalsooccuratlowanaestheticdosesinthedevelopingpig[42,43].Inthepresentstudy,somepigletsshowedtimespanswithBSupp,andthustheywereexcludedfromtheanalysis.Inapreviousstudyofjuvenilepigs,individualanimalsalsoexhibitedBSuppatisoﬂuraneconcentrationsofsub-MAClevels[44].Ingeneral,isoﬂuranedepressesthedose-relatedcorticalelectricalactivityinjuvenilepigs[42].However,theoccurrenceofmovementreactionstonoxiousstimulationinjuvenilepigsandratswithBSupphasbeenreported[37,45,46].4.1.EEGReactionPigletcastrationincludestwodistinctpainfulaspects,incisionofthescrotalskinandpullingandcuttingofthespermaticcords,whichissuggestedtobethemostpainfulcomponentofcastration[3,47].IntheEEGmeasurements,bothnoxiousstimuliwereanalysedseparately.ThecorticalreactionsintheNaClgroupwereexpectedtoreﬂecttheunalteredandmostintensenoxiousstimulusinthisexperimentbecausenoanalgesiawasapplied.Forthisgroup,abiphasicresponsetoboththeskinincisionandcuttingofthespermaticcordswasobservedthatwassimilarinshapetotheinterdigitalpinchresponse.TheEEGreactionwasalteredorstronglyattenuatedinthelocalanaestheticandhandlinggroups.Afterapriorapplicationofeitherlidocaine,mepivacaineorprocaine,theincisionofthescrotalskinevokednosigniﬁcantreactionsinthecerebralcortex,oratleastastronglyattenuatedreaction,inthebupivacainegroup.Followingthecuttingofthespermaticcords,ashortbutsigniﬁcantactivationwasobservedinthelidocainegroup,but Animals2022,12,2309 12of17 nosigniﬁcantchangesweredetectedintheotherlocalanaestheticgroups.ThecomparisonoftheintensechangesduringtheearlyandlateresponseobservedintheNaClgroupwiththatinthegroupsthatreceivedlocalanaestheticandthehandlinggroupalsosuggestsweakerresponsesinthelocalanaestheticgroupsandinthehandlinggroup.TheEEGreactiontonoxiousstimulationundergeneralanaesthesiamaybemani-fold[6].Inpatientsundergeneralanaesthesia,noxiousstimulationmaydecreaseEEGpowerinthealphaband[48,49]orincreasethepowerinthedeltaband[50]orbetaband[51].VariousEEGreactionstonoxiousstimulationthatmaydependonthespecies,age,thetypeanddoseofgeneralanaestheticused,thetypeandintensityofstimulationandtheamountofanalgesichavealsobeenreportedforinterventionsinanimals[8,11,12,44,52,53].TheobservedEEGreactiontotheinterdigitalpinchwithanearlydecreaseinslowoscillatoryactivityfollowedbyanincreaseinoscillatoryactivityabove10Hzmostlikelyindicatesachangetowardsarousal.Theadditionalstrongincreaseinpowerinthe10–15Hzrangeduringtheﬁrstsecondsafterthestimulusbeganmaybeattributedtotheclampthatwasstillattachedtotheskinfold.EEGarousalindicatesashiftinEEGactivityfromthelowertothehigherfrequencyrange.Thesehigherfrequenciesarisefromadesynchronisationofneuronalactivityandareusuallyassociatedwithhighercorticalfunction[54].ThearousalreactionmayberegardedasachangeintheEEGpatterninthedirectionoftheconsciousEEGpattern[17].Itisnotaspeciﬁcindicatorofnociceptiveinputbutrepresentsacommonreactiontonoxiousstimulationinhumansandhasalsobeendescribedinanimals[9,14,18].Hagaetal.[22]didnotobservesigniﬁcantEEGchangesindevelopingpigsfollowinginnocuousnoxiousmechanicalstimulationunderisoﬂuraneanaesthesia.Anunderlyingbiphasicresponsepatternasobservedinthepresentstudymayhavemaskedtheﬁndings.Otherﬁndingsseemtobesimilartothepresentresults.EEGdatarecordedinadultgoatsandaveragedover1minshowedthatthegoatsreactedtoaclamponthedewclawunderisoﬂuraneanaesthesiawithdecreasesintheprocessedparameterstotal,delta,thetaandalphapower,whilethebetapowerwasunchanged[10].Nevertheless,theresultsmaynotbedirectlycomparablewiththoseofthepresentstudyduetodifferencesinseveralparameters,e.g.,species,age,EEGsegmentlengthusedforanalysisandtheuseofprocessedEEGparameters.EEGmeasureswerealsousedtoexaminesurgicalnoxiousstimulationinducedbytaildockinginpiglets.AcomparisonofEEGresponsesinpigletsofeither2or20daysofageshowedaneffectofageontheEEGreaction[19].YoungpigletsshowedlittleEEGresponsetotaildocking,whereasolderpigletsreactedwithsigniﬁcantchangesinmedianfrequencyandtotalpower.Johnsonetal.alsodetecteddifferentEEGresponsesinpigletsbetween1and15daysofage[21].ThevaryingEEGreactionsofpigletsintheﬁrst20daysoflifeimplythatmajorchangesoccurintheEEGresponsesofpigletstonoxiousstimulation,particularlyduringtheﬁrstweeksoflife.BothstudiesdocumentedchangesinprocessedEEGparametersfollowingtaildockingunderhalothaneanaesthesia,butdetailedEEGdataaremissing.DifferentnoxiousstimulicausedifferingEEGchangesaswell[12].Therefore,acomparisonwiththepresentresultsisonlypossibleunderrestrictions.StudiesofEEGresponsestosurgicalcastrationinpigletsexist,althoughnoneofthesestudieswereperformedunderisofluraneanaesthesia.However,theanaestheticagentusediscrucialbecausedifferentanaestheticagentsinducedifferentEEGpatterns[34,53,55,56],andthechoiceofanaestheticagentmaypartiallyaccountfordifferencesinresultsbetweenEEGpatternsinnociceptivestudies[7].InitialfindingsfromWaldmannetal.[18]onthecastrationofpigletsalsorevealEEGarousalreactionsintheEEGsofindividualpigletsfollowingcastrationunderbarbiturateanaesthesia.HagaandRanheim[17]observeddecreasesindelta,theta,andalphabandsandtotalEEGpowerfollowingcastrationunderhalothaneanaesthesiawithoutadditionalanalgesiainapproximatelythree-week-oldpiglets.PigletsthatreceivedlidocaineeitherintrafunicularlyorintratesticularlyshowedalesspronouncedEEGresponse[17].Thepresentedresultstendtobesimilar,asreactionsinthecerebralcortexweredecreasedoratleastalteredafterlocalanaesthesiawasapplied.BecauseoftheuseofEEG Animals2022,12,2309 13of17 bandpowerandthelackofdetailedinformationregardingchangesintheEEGspectruminthesestudies,comparingthedetailedEEGresponsesisnotpossible.TheinjectionoflocalanaestheticsyieldedtheexpectedheterogeneousEEGreactions.ThedataindicatethatintratesticularandsubscrotalinjectionsprovokesigniﬁcantchangesinEEGwaves,irrespectiveofwhichﬂuidisused.Thereactionsdifferedinallgroupsinregardstothebiphasicreactionpatternobservedfollowingtheinterdigitalpinch,ornoxiousstimulationbyskinincisionanddissectionofthespermaticcordwhennolocalanaestheticswerecoapplied.ThevaryingpharmacologicalpropertiesofthelocalanaestheticsmaycausedifferentstimulithatarenotuniformandmightexplainthevariouspostinjectionEEGpatterns.Nonetheless,thedatasetsfromtheinjectionsareconsideredvaluableandarepresentedassupplementaldata(FigureS1).ForthepresentationoftheEEGresults,thepowerspectrumusedforthegenerationoftheDSAplotsfrom5sEEGepisodeswitha1soverlapwascalculatedwiththegoalofnotmissinganyEEGreactionswithashortduration.Althoughthep<0.1thresholdisunusual,itmayhelpusbetteridentifythepossibleresponse;however,duetothelimitedsamplesize,theﬁndingsfromthedifferentgroupsarepreliminaryresults.Thestudyrefrainsfromdiscussingscatteredpixelsbecauseoftheincreasedriskofdiscussingfalsepositivesduetomultiplecomparisons,whichiswhytheclusterapproachwasused.Werefrainedfromfocusingonchangesinpredeﬁnedfrequencybandsorinparametersusinginformationfromtheentirespectrum.First,thedivisionoftheEEGpowerspectrumofanimalsintothefrequencybandsalpha,beta,deltaandthetamaybequitearbitrary[7],andvariabilityhasbeenobservedinthespeciﬁcfrequencyrangethatdeﬁneseachband[57].Second,processedparameters,suchasthemedianfrequencyorwidefrequencybands(e.g.,thebetarangefrom~12–30Hz),mightmaskeffects.Forinstance,anincreaseinpowerfrom12–16Hzandasimultaneousdecreaseinpowerfrom20–25Hzwouldnothaveaneffectonthebetaband.Ingeneral,differencesintheanaestheticagentused,noxiousstimuli,species,ageoftheanimals,evaluatedparameters,andtimescalespreventacomparisonofstudies.ThisstudypresentstheunprocessedspectralresponseintheEEG,whichmayfacilitatethetranslationoftheﬁndingstotheresultsfromotherexperiments.TheuseofDSAplotsandthedecisiontorefrainfromusingprocessedEEGparametersallowedustoobservechronologicalchangesinEEGactivitypatternsovertheentirefrequencyrange.ThisapproachdescribestheEEGreactionsinamoredetailedmannerthanusingprocessedEEGparametersorbandpowersderivedfromthespectrum.4.2.FOSProteinTheexpressionoftheimmediateearlygenec-FOSanditsproteinproductFOSinneuronsofthespinalcordisawell-establishedmarkertoquantifyneuralactivityinresponsetonoxiousstimulation[25,26].Noxiousperipheralstimulationevokesarapidchangeingeneexpressionofthepostsynapticneuronsinthedorsalhornofthespinalcord[24].ThemRNAoftheimmediateearlygenec-FOSisdetectedwithinfiveminutesfollowingphysicalstress,suchasinjury[58].Thec-FOSgeneencodesthenuclearproteinFOS,whichisdetectedinthesameneuronswithaproteinpeakobservedatapproximately90–120minaftertheinductionofgeneexpression[23,25,59].Afternoxiousperipheralstimulation,immunohistochemicalFOS-positiveneuronsarepredominantlyobservedinlaminaeIandIIofthedorsalhorn,alongwithsomelabellinginlaminaeIII-VI[26,27].AsemiquantitativeapproachwasusedtoevaluateFOSexpressioninthespinaldorsalhorn,whichincludeslaminaeI–VI.Thisapproachhasnotbeenvalidatedforuseasastand-aloneread-outofneuronalstimulationinthespinalcord.However,incombinationwiththeEEGresults,itservesasanadditionalparameter,whichcanbeusedtofurtherevaluatethenociceptiveresponse.Themajorityofafferentneuronssupplyingtheporcinetestisandadjacentstructuresarelocatedindorsalrootgangliaoflumbarsegmentsonetothreeandsacralsegmentsonetothree[60–62].Therefore,thesespinalcordsegmentswereincludedintheanalysis.SigniﬁcantlylowerlevelsofFOSexpressionweredetectedinthenucleiofspinalneurons Animals2022,12,2309 14of17 inthepigletsthatwerecastratedafterapplicationofalocalanaesthetic,exceptforthebupivacainegroup,andinanimalsthatwereonlyhandled,comparedwithanimalsun-dergoingcastrationwithoutanalgesia.FOSexpressionwasadditionallyanalysedinonlytheanimalsthatdidnotshowBSuppthroughouttheexperimenttoevaluateapossibleeffectoftheanaestheticlevel(i.e.,BSupp).Theresultsweresimilarandarepresentedinthesupportinginformation(TableS2).TheFOSresultssupportdatafromastudybyNyborgetal.,whoreportedalowernumberofFOS-positiveneuronsinthespinaldorsalhorninpigletsundergoingcastrationwithlocalanaesthesiathaninpigletsundergoingsurgerywithoutanalgesia[29].FOSexpressioninthedorsalhornofpigletswasalsoreducedaftercastrationunderCO2anaesthesia[28].Additionally,localanaesthesiawaseffectiveatreducingthenumberofFOS-positiveneuronsinjuvenilepigsundergoinglaparotomywhencomparedwithanimalsundergoingsurgerywithoutanaesthesia[27].Inthepresentexperiment,allanimalsshowedatleastslightstaininginsingleneu-rons.ThisﬁndingmayresultfromthefactthatFOSexpressioninthisexperimentrep-resentsthecumulativeresponsetonoxiousstimulationoccurringinthetimeperiodof~90–120minbeforeharvestingthespinalcordandtransferredvianeuronsinL1,L2,L3andS1-3.Allanimalsexperiencedatleasttwointerdigitalpinches,thesecondofwhichwasappliedfourminutesfollowingspermaticcorddissectionandthereforeoccurredintherelevanttimeframe.Duetothesimilarinnervationoftheinterdigitalareaofthehindclaw,theinterdigitalpinchesverylikelycontributedtoFOSexpressioninthepresentstudy.Moreover,changesinheartrateandmeanarterialbloodpressureinthehandlinggroupwereobservedfollowinginjection,skinincisionandspermaticcorddissection[3].Obviously,thetouchingandﬁxationofthetestesalsoledtoadversereactions,whichpossiblyalsocontributedtothebaselineFOSexpression.DespitebaselineFOSexpression,signiﬁcantdifferencesinFOSexpressionwereevidentbetweentheanimalstreatedwithoutanalgesiaandanimalstreatedwithlocalanaesthesiaoronly-handledanimals.Therefore,weassumedthatFOSexpressionmaybeusedasanindicatorofnociceptionduringsuck-lingpigletcastration.However,furtherstudieswithlargeranimalnumberswouldbebeneﬁcialtoconﬁrmthisﬁnding.OnelimitationofthestudyisthatasamplesizecalculationfortheoverallstudywasnotperformedbasedonEEGeffectsorFOSexpression.Haga,TevikandMoerch[22]foundbloodpressuretobeamoresensitiveindicatorofnociceptioninjuvenilepigsthanelectroen-cephalographicparameters.Therefore,thesamplesizeestimationwasspecificallybasedonthecorrespondinghaemodynamicparameters.Hence,thepowerofthepresentstudymaybetoolowtodrawdefiniteconclusionsbasedonEEGandFOSmeasurementsasstand-aloneparametersregardingtheefficacyofthefourlocalanaestheticsforpigletcastrationortocom-parethemwitheachother.Furtherstudieswithalargersamplesizeareneededtodescribetheeffectsonthelocalanaestheticgroupsinmoredetailandtocomparethegroups.5.ConclusionsTheEEGreactionsof3-to7-day-oldpigletstoaninnocuousinterdigitalpinchandtosurgicalcastrationunderlightisofluraneanaesthesiaweretracked.BoththenoxiousstimulationcausedbyaninterdigitalpinchandbycastrationwithoutpainreliefwerevisibleintheEEGasabiphasic‘early’and’late’response.ThisEEGreactionpatternwasattenuatedintheanimalsthatreceivedlocalanaesthesiaandinanimalsofthehandlinggroup.WeareconfidentinouridentificationofthenociceptiveresponseintheEEGbecauseofthe(i)repetitivenatureofthesignalsobservedfollowinginterdigitalpinchingandsurgicalinterventionswithoutanalgesia,(ii)theattenuatedresponsetosurgicalinterventionwithlocalanaesthetics,and(iii)thereducedFOSexpressioninanimalstreatedwithlocalanaesthetics.Itcanbeassumedthattheuseoflocalanaestheticsinpigletsundergoingsurgicalcastrationunderisoﬂuraneanaesthesiaverylikelyattenuatesnociceptivetransmissionunderstandardisedlaboratoryconditions.TheEEGandFOSresultsareconsistentwiththesigniﬁcantlyreducedheartrateandbloodpressureandthedecreasedmovementreactionsthatweredetectedinthe Animals2022,12,2309 15of17 sameanimals[3].TheresultsoftheEEGandFOSmeasurementsincombinationwiththecorrespondingresultsforthehaemodynamicparametersandtheevaluationoflimbmovements[3]providevaluableinformationwithwhichtoformanoverallunderstandingofthenociceptiveresponseofpigletstocastrationwithorwithoutlocalanaesthetics.Additionally,theresultsfromtheEEGandFOSanalysesprovidestrongpilotdataforfurtherstudiesregardingEEGnociceptioninpiglets.Toourknowledge,thedetailedEEGreactionsofpigletstoanoxiousinterdigitalpinchandsurgicalcastration,aswellasadescriptionofhowlocalanaestheticsmayalterthispattern,havenotbeenpresentedtodate.Furtherinvestigationsarerequiredtodeterminewhetherorhowtheresultsrelatetopainperceptioninconsciouspigletsandunderﬁeldstudyconditions.SupplementaryMaterials:Thefollowingsupportinginformationcanbedownloadedathttps://www.mdpi.com/article/10.3390/ani12182309/s1,SupplementaryMaterial1:FigureS1:EEGChangesafterIntratesticularandSubscrotalInjections;Supplementarymaterial2:FOSProteinAnalysisinOnlyAnimalswithoutBSupp;FigureS2:ScatterPlotoftheFOSStainingScore(BasedonAnimalswithoutBSupp);andTableS2:NumberofAnimalsIncludedintheFOSProteinAnalysis(BasedonAnimalswithoutBSupp).AuthorContributions:Conceptualisation,S.Z.andC.B.;Datacuration,J.R.andM.K.;Formalanalysis,M.K.;Fundingacquisition,S.Z.andC.B.;Investigation,J.R.,J.W.,A.M.S.,J.F.,S.S.,P.D.,N.A.,T.G.,A.G.andR.M.;Methodology,J.R.,J.W.,A.M.S.,J.F.,S.S.,P.D.,N.A.,T.G.andK.S.;Projectadministration,S.Z.andC.B.;Resources,M.R.,G.S.andC.B.;Supervision,S.B.,M.H.E.,M.R.,S.Z.,G.S.andC.B.;Validation,J.R.,M.K.andK.S.;Visualisation,J.R.,M.K.andK.S.;Writing—originaldraft,J.R.andM.K.;Writing—review&editing,J.W.,A.M.S.,J.F.,S.S.,P.D.,N.A.,T.G.,A.G.,R.M.,S.B.,M.H.E.,M.R.,S.Z.,G.S.,K.S.andC.B.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:ThisresearchwasfundedbytheGermanFederalMinistryofFoodandAgriculture(BMEL,https://www.bmel.de)basedonadecisionoftheParliamentoftheFederalRepublicofGermany,grantedbytheFederalOfﬁceforAgricultureandFood(BLE,https://www.ble.de,grantnumber:2817HS016,2817HS013).InstitutionalReviewBoardStatement:ThestudywasperformedincompliancewiththeEUDirec-tive2010/63/EUforanimalexperimentsandtheGermanAnimalWelfareAct[32].AllprocedureswereapprovedbytheEthicsCommitteeforAnimalExperimentsoftheGovernmentofUpperBavaria,Munich,Germany(ReferenceNumberROB-55.2-2532.Vet_02-19-11).InformedConsentStatement:Notapplicable.DataAvailabilityStatement:Notapplicable.Acknowledgments:TheauthorswouldliketothankStephanKratzerandSebastianBerger,whosupportedusinestablishingtheelectroencephalographicmeasurements;EvaPrötzl,whohelpedtoprovidetheMATLABcodefortheclusteranalysis;MarionMielkeandOlgaSeelbach,whoperformedtheparafﬁnembeddingandcutting,aswellastheimmunohistologicalstaining;andJohannaBrandl,whoprovidedsigniﬁcantsupportinthepreparationofthispaper.ConﬂictsofInterest:Theauthorshavenoconﬂictsofinteresttodeclare.References1.Hansson,M.;Lundeheim,N.;Nyman,G.;Johansson,G.Effectoflocalanaesthesiaand/oranalgesiaonpainresponsesinducedbypigletcastration.ActaVet.Scand.2011,53,34.[CrossRef]2.Kluivers,M.;Hopster,H.;Spoolder,H.A.M.CastrationunderAnaesthesiaand/orAnalgesiainCommercialPigProduction;AnimalSciencesGroup:Lelystad,TheNetherlands,2007.3.Saller,A.M.;Werner,J.;Reiser,J.;Senf,S.;Deffner,P.;Abendschön,N.;Weiß,C.;Fischer,J.;Schörwerth,A.;Miller,R.;etal.Localanesthesiainpigletsundergoingcastration-Acomparativestudytoinvestigatetheanalgesiceffectsoffourlocalanestheticsonthebasisofacutephysiologicalresponsesandlimbmovements.PLoSONE2020,15,e0236742.[CrossRef][PubMed]4.Rose,J.;Arlinghaus,R.;Cooke,S.;Diggles,D.B.K.;Sawynok,W.;Stevens,D.;Wynne,C.D.L.Canﬁshreallyfeelpain?FishFish.2013,15,97–133.[CrossRef]5.Mathews,K.;Kronen,P.W.;Lascelles,D.;Nolan,A.;Robertson,S.;Steagall,P.V.;Wright,B.;Yamashita,K.Guidelinesforrecognition,assessmentandtreatmentofpain:WSAVAGlobalPainCouncilmembersandco-authorsofthisdocument.J.SmallAnim.Pract.2014,55,E10–E68.[CrossRef][PubMed] 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6.García,P.S.;Kreuzer,M.;Hight,D.;Sleigh,J.W.Effectsofnoxiousstimulationontheelectroencephalogramduringgeneralanaesthesia:Anarrativereviewandapproachtoanalgesictitration.Br.J.Anaesth.2021,126,445–457.[CrossRef][PubMed]7.Murrell,J.C.;Johnson,C.B.Neurophysiologicaltechniquestoassesspaininanimals.J.Vet.Pharmacol.Ther.2006,29,325–335.[CrossRef][PubMed]8.Otto,K.A.Effectsofaveragingdataseriesontheelectroencephalographicresponsetonoxiousvisceralstimulationinisoﬂurane-anaesthetizeddogs.Res.Vet.Sci.2007,83,385–393.[CrossRef][PubMed]9.Otto,K.A.;Mally,P.NoxiousstimulationduringorthopaedicsurgeryresultsinEEG‘arousal’or‘paradoxicalarousal’reactioninisoﬂurane-anaesthetisedsheep.Res.Vet.Sci.2003,75,103–112.[CrossRef]10.Antognini,J.F.;Carstens,E.Isoﬂuranebluntselectroencephalographicandthalamic-reticularformationresponsestonoxiousstimulationingoats.Anesthesiology1999,91,1770–1779.[CrossRef]11.Johnson,C.B.;Sylvester,S.P.;Stafford,K.J.;Mitchinson,S.L.;Ward,R.N.;Mellor,D.J.Effectsofageontheelectroencephalographicresponsetocastrationinlambsanaesthetizedwithhalothaneinoxygenfrombirthto6weeksold.Vet.Anaesth.Analg.2009,36,273–279.[CrossRef]12.Murrell,J.C.;Mitchinson,S.L.;Waters,D.;Johnson,C.B.Comparativeeffectofthermal,mechanical,andelectricalnoxiousstimuliontheelectroencephalogramoftherat.Br.J.Anaesth.2007,98,366–371.[CrossRef]13.Orth,M.;Barter,L.;Dominguez,C.;Atherley,R.;Carstens,E.;Antognini,J.Halothaneandpropofoldifferentiallyaffectelectroencephalographicresponsestonoxiousstimulation.Br.J.Anaesth.2005,95,477–484.[CrossRef][PubMed]14.Murrell,J.C.;Johnson,C.B.;White,K.L.;Taylor,P.M.;Haberham,Z.L.;Waterman-Pearson,A.E.ChangesintheEEGduringcastrationinhorsesandponiesanaesthetizedwithhalothane.Vet.Anaesth.Analg.2003,30,138–146.[CrossRef][PubMed]15.Haga,H.A.;Dolvik,N.I.Electroencephalographicandcardiovascularvariablesasnociceptiveindicatorsinisoﬂurane-anaesthetizedhorses.Vet.Anaesth.Analg.2005,32,128–135.[CrossRef][PubMed]16.Diesch,T.J.;Mellor,D.J.;Johnson,C.B.;Lentle,R.G.Electroencephalographicresponsestotailclampinginanaesthetizedratpups.Lab.Anim.2009,43,224–231.[CrossRef]17.Haga,H.A.;Ranheim,B.Castrationofpiglets:Theanalgesiceffectsofintratesticularandintrafunicularlidocaineinjection.Vet.Anaesth.Analg.2005,32,1–9.[CrossRef]18.Waldmann,V.;Otto,K.H.;Bollwahn,W.Pigletcastration-painsensationandpainelimination.Dtsch.Tierarztl.Wochenschr.1994,101,105–109.19.Kells,N.J.;Beausoleil,N.J.;Chambers,J.P.;Sutherland,M.A.;Morrison,R.S.;Johnson,C.B.Electroencephalographicresponsesofanaesthetizedpigs(Susscrofa)totaildockingusingclippersorcauteryironperformedat2or20daysofage.Vet.Anaesth.Analg.2017,44,1156–1165.[CrossRef]20.Kells,N.J.;Beausoleil,N.J.;Sutherland,M.A.;Morrison,R.M.;Johnson,C.B.Electroencephalographicassessmentoforalmeloxicam,topicalanaestheticcreamandcauteryironformitigatingacutepaininpigs(Susscrofa)undergoingtaildocking.Vet.Anaesth.Analg.2017,44,1166–1174.[CrossRef]21.Johnson,C.;Sutherland,M.;Beausoleil,N.;Kells,N.ValidationofEEGMeasuresforPainAssessmentinPigletsAged0to10Days.pork.org.NPB#C-13–188;MasseyUnivesity:PalmerstonNorth,NewZealand,2015.22.Haga,H.A.;Tevik,A.;Moerch,H.Electroencephalographicandcardiovascularindicatorsofnociceptionduringisoﬂuraneanaesthesiainpigs.Vet.Anaesth.Analg.2001,28,126–131.[CrossRef]23.Munglani,R.;Hunt,S.P.Molecularbiologyofpain.Br.J.Anaesth.1995,75,186–192.[CrossRef]24.Hunt,S.P.;Pini,A.;Evan,G.Inductionofc-fos-likeproteininspinalcordneuronsfollowingsensorystimulation.Nature1987,328,632–634.[CrossRef][PubMed]25.Harris,J.A.Usingc-fosasaneuralmarkerofpain.BrainRes.Bull.1998,45,1–8.[CrossRef]26.Coggeshall,R.E.Fos,nociceptionandthedorsalhorn.Prog.Neurobiol.2005,77,299–352.[CrossRef][PubMed]27.Lykkegaard,K.;Lauritzen,B.;Tessem,L.;Weikop,P.;Svendsen,O.LocalanaestheticsattenuatesspinalnociceptionandHPA-axisactivationduringexperimentallaparotomyinpigs.Res.Vet.Sci.2005,79,245–251.[CrossRef][PubMed]28.Svendsen,O.Castrationofpigletsundercarbondioxide(CO2)anaesthesia.J.Vet.Pharmacol.Ther.2006,29,54–55.[CrossRef]29.Nyborg,P.Y.;Sørig,A.;Lykkegaard,K.;Svendsen,O.Nociceptionaftercastrationofjuvenilepigsdeterminedbyquantitativeestimationofc-Fosexpressingneuronsinthespinalcorddorsalhorn.DanskVeterinærtidsskrift2000,83,16–17.30.Ruggiero,D.A.;Sica,A.L.;Anwar,M.;Frasier,I.;Gootman,N.;Gootman,P.M.Inductionofc-fosgeneexpressionbyspinalcordtransectioninSusscrofa.BrainRes.1997,759,301–305.[CrossRef]31.Werner,J.;Saller,A.;Reiser,J.;Senf,S.;Deffner,P.;Abendschön,N.;Fischer,J.;Grott,A.;Miller,R.;Zablotski,Y.;etal.EvaluationofTwoInjectionTechniquesinCombinationwiththeLocalAnestheticsLidocaineandMepivacaineforPigletsUndergoingSurgicalCastration.Animals2022,12,1028.[CrossRef]32.Tierschutzgesetz.TierschutzgesetzinderFassungderBekanntmachungvom18.Mai2006(BGBl.IS.1206,1313),daszuletztdurchArtikel105desGesetzesvom10.August2021(BGBl.IS.3436)geändertwordenist.2021.Availableonline:https://www.gesetze-im-internet.de/tierschg/BJNR012770972.html(accessedon31August2022).33.Sassenhagen,J.;Draschkow,D.Cluster-basedpermutationtestsofMEG/EEGdatad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35.Kreuzer,M.;Stern,M.A.;Hight,D.;Berger,S.;Schneider,G.;Sleigh,J.W.;Garcia,P.S.Spectralandentropicfeaturesarealteredbyageintheelectroencephalograminpatientsundersevoﬂuraneanesthesia.Anesthesiology2020,135,1003–1016.[CrossRef]36.Hentschke,H.;Stüttgen,M.C.Computationofmeasuresofeffectsizeforneurosciencedatasets.Eur.J.Neurosci.2011,34,1887–1894.[CrossRef]37.Haga,H.A.;Tevik,A.;Moerch,H.Motorresponsestostimulationduringisoﬂuraneanaesthesiainpigs.Vet.Anaesth.Analg.2002,29,69–75.[CrossRef]38.Satas,S.;Haaland,K.;Thoresen,M.;Steen,P.A.MACforhalothaneandisoﬂuraneduringnormothermiaandhypothermiainthenewbornpiglet.ActaAnaesthesiol.Scand.1996,40,452–456.[CrossRef]39.Arras,M.;Autenried,P.;Rettich,A.;Spaeni,D.;Rülicke,T.Optimizationofintraperitonealinjectionanesthesiainmice:Drugs,dosages,adverseeffects,andanesthesiadepth.Comp.Med.2001,51,443–456.40.LeBars,D.;Gozariu,M.;Cadden,S.W.Animalmodelsofnociception.Pharmacol.Rev.2001,53,597–652.41.Rintisch,U.;Baars,J.;Lahrmann,K.H.Evaluationofperioperativeanalgesiabynociceptiveﬂexorreﬂexinpigsunderketamine-azaperone-generalanaesthesia.Berl.Munch.Tierarztl.Wochenschr.2012,125,96–102.42.Rampil,I.J.;Weiskopf,R.B.;Brown,J.G.;Eger,E.I.,2nd;Johnson,B.H.;Holmes,M.A.;Donegan,J.H.I653andisoﬂuraneproducesimilardose-relatedchangesintheelectroencephalogramofpigs.Anesthesiology1988,69,298–302.[CrossRef]43.Niedermeyer,E.;Sherman,D.L.;Geocadin,R.J.;Hansen,H.C.;Hanley,D.F.Theburst-suppressionelectroencephalogram.Clin.Electroencephalogr.1999,30,99–105.[CrossRef]44.Haga,H.A.;Ranheim,B.;Spadavecchia,C.Effectsofisoﬂuraneuponminimumalveolarconcentrationandcerebralcortexdepressioninpigsandgoats:Aninterspeciescomparison.Vet.J.2011,187,217–220.[CrossRef]45.Orth,M.;Bravo,E.;Barter,L.;Carstens,E.;Antognini,J.F.Thedifferentialeffectsofhalothaneandisoﬂuraneonelectroencephalo-graphicresponsestoelectricalmicrostimulationofthereticularformation.AnesthAnalg.2006,102,1709–1714.[CrossRef]46.Rampil,I.J.;Laster,M.J.Nocorrelationbetweenquantitativeelectroencephalographicmeasurementsandmovementresponsetonoxiousstimuliduringisoﬂuraneanesthesiainrats.Anesthesiology1992,77,920–925.[CrossRef]47.Taylor,A.A.;Weary,D.M.Vocalresponsesofpigletstocastration:Identifyingproceduralsourcesofpain.Appl.Anim.Behav.Sci.2000,70,17–26.[CrossRef]48.Hight,D.F.;Gaskell,A.L.;Kreuzer,M.;Voss,L.J.;García,P.S.;Sleigh,J.W.Transientelectroencephalographicalphapowerlossduringmaintenanceofgeneralanaesthesia.Br.J.Anaesth.2019,122,635–642.[CrossRef][PubMed]49.Hagihira,S.;Takashina,M.;Mori,T.;Ueyama,H.;Mashimo,T.Electroencephalographicbicoherenceissensitivetonoxiousstimuliduringisoﬂuraneorsevoﬂuraneanesthesia.J.Am.Soc.Anesthesiol.2004,100,818–825.[CrossRef]50.Kochs,E.;Bischoff,P.;Pichlmeier,U.;Schulte,A.E.J.Surgicalstimulationinduceschangesinbrainelectricalactivityduringisoﬂurane/nitrousoxideanesthesia.Atopographicelectroencephalographicanalysis.Anesthesiology1994,80,1026–1034.[CrossRef]51.Rundshagen,I.;Schröder,T.;Heinze,J.;Prichep,L.;John,E.R.;Kox,W.J.Topographicelectroencephalography:Endotrachealintubationduringanaesthesiawithpropofol/fentanyl.Anasthesiol.Intensivmed.Notfallmed.Schmerzther.2005,40,633–639.[CrossRef]52.Ekström,P.M.;Short,C.E.;Geimer,T.R.Electroencephalographyofdetomidine-ketamine-halothaneanddetomidine-ketamine-isoﬂuraneanesthetizedhorsesduringorthopedicsurgery.Acomparison.Vet.Surg.1993,22,414–418.[CrossRef]53.Murrell,J.C.;Waters,D.;Johnson,C.B.Comparativeeffectsofhalothane,isoﬂurane,sevoﬂuraneanddesﬂuraneontheelectroen-cephalogramoftherat.Lab.Anim.2008,42,161–170.[CrossRef]54.Rampil,I.J.AprimerforEEGsignalprocessinginanesthesia.Anesthesiology1998,89,980–1002.[CrossRef]55.Akeju,O.;Pavone,K.J.;Westover,M.B.;Vazquez,R.;Prerau,M.J.;Harrell,P.G.;Hartnack,K.E.;Rhee,J.;Sampson,A.L.;Habeeb,K.;etal.Acomparisonofpropofol-anddexmedetomidine-inducedelectroencephalogramdynamicsusingspectralandcoherenceanalysis.Anesthesiology2014,121,978–989.[CrossRef]56.Johnson,C.B.;Taylor,P.M.Comparisonoftheeffectsofhalothane,isoﬂuraneandmethoxyﬂuraneontheelectroencephalogramofthehorse.Br.J.Anaesth.1998,81,748–753.[CrossRef]57.Newson,J.J.;Thiagarajan,T.C.EEGFrequencyBandsinPsychiatricDisorders:AReviewofRestingStateStudies.Front.Hum.Neurosci.2018,12,521.[CrossRef]58.Reiner,G.;Heinricy,L.;Brenig,B.;Geldermann,H.;Dzapo,V.Cloning,structuralorganization,andchromosomalassignmentoftheporcinec-fosproto-oncogene,FOS.Cytogenet.GenomeRes.2000,89,59–61.[CrossRef]59.Leah,J.D.;Sandkuhler,J.;Herdegen,T.;Murashov,A.;Zimmermann,M.PotentiatedexpressionofFOSproteinintheratspinalcordfollowingbilateralnoxiouscutaneousstimulation.Neuroscience1992,48,525–532.[CrossRef]60.Sienkiewicz,W.Sourcesoftheporcinetestisinnervation.Andrologia2010,42,395–403.[CrossRef]61.Nickel,R.;Schummer,A.;Seiferle,E.Nervensystem,sinnesorgane,endokrinedrüsen.InLehrbuchderAnatomiederHaustiere;PareyVerlag:Stuttgart,Germany,1992;p.273.62.Kaleczyc,J.;Scheuermann,D.W.;Pidsudko,Z.;Majewski,M.;Lakomy,M.;Timmermans,J.P.Distribution,immunohistochemicalcharacteristicsandnervepathwaysofprimarysensoryneuronssupplyingthe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aAbendschön3,TanjaGroll4 ,AndreaGrott5,ReginaMiller5,ShanaBergmann5,MichaelH.Erhard5 ,MathiasRitzmann3,SusanneZöls3,GerhardSchneider2 ,KatjaSteiger4,† andChristineBaumgartner1,*,†1CenterofPreclinicalResearch,TechnicalUniversityofMunich,81675Munich,Germany2DepartmentofAnesthesiologyandCriticalCare,SchoolofMedicine,TechnicalUniversityofMunich,81675Munich,Germany3ClinicforSwine,CenterforClinicalVeterinaryMedicine,LMUMunich,85764Oberschleissheim,Germany4InstituteofPathology,SchoolofMedicine,TechnicalUniversityofMunich,81675Munich,Germany5ChairofAnimalWelfare,Ethology,AnimalHygieneandHusbandry,LMUMunich,80539Munich,Germany*Correspondence:christine.baumgartner@tum.de†Theseauthorscontributedequallytothiswork.SimpleSummary:Efﬁcientanalgesiaduringsurgicalpigletcastrationisanimportantanimalwelfareissue.Thepresentstudyispartofalargerstudydesignedtoinvestigatetheefﬁcacyoffourlocalanaesthetics(bupivacaine,lidocaine,mepivacaineandprocaine)foranalgesiaduringcastration.Inconsciouspiglets,itisdifﬁculttodistinguishbetweenstress,e.g.,duetohandling,andpainduringcastration.Therefore,thisstudywasconductedunderlightisoﬂuraneanaesthesia.Thelevelofanaesthesiawasadjustedsuchthatmovementreactionstoaperipheralnociceptivestimulusstilloccurred.Wepresenttheresultsoftheinvestigationoftwopossibleparametersforthedetectionofnociception:thereactionpatternintheelectroencephalogram(EEG)andFOSproteinexpressioninthespinaldorsalhorn.Intheelectroencephalogram,abiphasicreactionpatterninresponsetonoxiousstimulationwasdetectedthatwasattenuatedoralteredbytheapplicationoflocalanaes-thetics.FOSexpression,whichwasexaminedpostmortem,wasdecreasedaftertheadministrationoflocalanaesthetics,exceptforbupivacaine.Basedontheseresults,localanaesthesiadecreasesnociceptivetransmissionduringpigletcastrationinthisexperimentalsetup.Whencombinedwiththecorrespondinghaemodynamicparametersandtheevaluationofdefensivemovementspresentedelsewhere,anoverallunderstandingofthenociceptiveresponsetocastrationcanbegenerated.Abstract:TheobjectiveofthisstudywastoinvestigatetheelectroencephalographicreactionpatternandFOSproteinexpressioninmalepigletsundergoingsurgicalcastrationunderlightisoﬂuraneanaesthesiawithorwithoutlocalanaesthesia.Theexperimentwasconductedunderisoﬂuraneanaesthesiatoexcludetheeffectoftheaffectivecomponentsofpainonthemeasurements.Changesintheoscillatoryactivityofthecerebralcortexovera90speriodafternoxiousstimulationorsim-ulatedinterventionswereanalysed.FOSexpressionwasdeterminedpostmortembyperformingimmunohistochemistryinthedorsalhornofthespinalcord.Theanalysisoftheresponsetoaninterdigitalpinchrevealedabiphasicreactionpatternintheelectroencephalogram(EEG)thatsim-ilarlywasobservedforthesurgicalstimuliduringthecastrationprocedureinthegroupwithoutanalgesia.ThisEEGresponsewasattenuatedoralteredbytheapplicationoflocalanaesthetics.ImmunohistochemicalstainingforFOSindicatedalowerexpressioninthehandlingandinthreelocalanaestheticgroupsthanintheanimalscastratedwithoutpainrelief.TheﬁndingsindicatethatEEGandFOSexpressionmayserveasindicatorsfornociceptioninpigletsunderlightisoﬂuraneanaesthesia.Aloweractivationofnociceptivepathwaysoccursduringcastrationaftertheappli-cationoflocalanaesthetics.However,EEGandFOSanalysesshouldbecombinedwithadditionalparameterstoassessnociception,e.g.,haemodynamicmonitoring.

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ertheappli-cationoflocalanaesthetics.However,EEGandFOSanalysesshouldbecombinedwithadditionalparameterstoassessnociception,e.g.,haemodynamicmonitoring. Animals2022,12,2309.https://doi.org/10.3390/ani12182309https://www.mdpi.com/journal/animals

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Animals2022,12,2309 2of17 Keywords:EEG;FOS;piglet;electroencephalography;castration;localanaesthesia;nociception;lidocaine;procaine;mepivacaine;bupivacaine;spectrogram;animal

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7 Keywords:EEG;FOS;piglet;electroencephalography;castration;localanaesthesia;nociception;lidocaine;procaine;mepivacaine;bupivacaine;spectrogram;animal 1.IntroductionSurgicalcastrationofmalepigletsoflessthaneightdaysofagewithoutanaesthesiahasbeenacommonpracticefordecadesinseveralcountriestopreventboar-taintedmeatandaggressivebehaviourofintactmalepigs,aswellastoobtaintherequestedmeatquality.However,surgicalcastrationwithoutpainreliefisapainfulintervention[1–3].Signiﬁcanteffortshavebeendevotedtodevelopingmethodsthatprovideeffectiveanalgesiaforthesurgicalcastrationofpigletsandtechniquesfortheexaminationofpainandnociceptionduringthisprocedure.Painisacomplexmultidimensionalexperienceinvolvingsensoryandaffective(emo-tional)componentsthatisonlyexperiencedbyconsciousanimalsandisasubjectiveemotion[4,5].Thepresentstudyaimedtomeasurenociception,thesensorycomponentofpain,andtoassesstheefﬁcacyoflocalanaestheticsduringthecastrationofmalepiglets.Anaesthesiawithisoﬂuranewasadministeredtoexcludetheeffectsofaffectivecompo-nentsonthemeasurements.Thus,theeffectsoffearandstressonmodulatingthepainexperienceduetotheconsciousexperienceofhandling,ﬁxationornoiseswereeliminated.Asisoﬂuranehasnoornegligibleanalgesicproperties,anociceptiveresponsewasstillpresent.Weaimedtoestablishadepthofanaesthesiaatwhichthenociceptivewithdrawalreﬂexwasstillpresent.EEGandFOSexpressionwereevaluatedattwodifferentlocationsinthenociceptivepathway:FOSexpressionwasmeasuredinthedorsalhornofthespinalcord,whichistheﬁrstrelaystationinnociceptivetransmissionofperipheralstimuli,andpain-inducedchangesincortical(electrical)activitywererecordedusinganEEG,representingtheﬁnalstageofnociceptivesignalling—thebrain.EEG,adirectmeasureofbrainelectricalactivity,enablestheimmediateassessmentofpossiblepain-inducedchanges.However,theappropriateidentiﬁcationofEEGchangescausedbynoxiousstimulationisdifﬁcultduetotheheterogeneityofpossibleEEGre-sponses[6,7].NumerousstudieshaveevaluatedchangesintheEEGsofanimalscausedbynoxiousstimulationundergeneralanaesthesia[8–16].Inpiglets,EEGmeasurementshavealsobeenconductedtoinvestigatethecorticalresponsestonoxiousstimulation.TheresultsdescribeEEGchangesfollowingcastration[17,18],exposuretoothersurgicalstimuli[19–21]andinnocuousnoxiousstimuli[22].Somestudiesinanimalshaverevealedreactions,whileothersfailedtoidentifychangesinEEGparametersfollowingnoxioussurgicalstimulationundergeneralanaesthesia.AllofthesestudiesusedprocessedEEGparameters,suchastotalpowerorspectraledgefrequencies,derivedfromtheEEGpowerspectrumtodescribetheirﬁndings.However,themereuseoftheseprocessedparameterscomplicatestheabilitytocompareﬁndingsfromdifferentstudies.Therefore,thegeneralspectralEEGresponsetonoxiousstimulationispresentedinadetailedmannerasdensityspectralarrays,anapproachthatdoesnotreduceinformationduetotheuseofprocessedEEGparameters.Peripheralnoxiousstimulationevokestherapidexpressionoftheimmediateearlygenec-FOSanditsproteinproductFOSinthenucleiofpostsynapticneuronsinthespinaldorsalhorn[23,24].Theexpressionofc-FOSandFOSinneuronsofthespinalcordisanestablishedmarkertoquantifyneuralactivityinresponsetonoxiousstimulationpostmortem[25,26].Itwasappliedinpigsaswell,andsinglestudieshaveevaluatedFOSexpressiontoinvestigatepainornociception,respectively,followingpigletcastration[27–31].Thegroupwithoutfurtheranalgesictreatment(NaCl)wascomparedwiththeanimalsthatreceivedlocalanaesthesiawitheither0.5%bupivacaine,2%lidocaine,2%mepivacaineor4%procaine,aswellaswiththeanimalsthatunderwentonlysimulatedinterventionstocorrelatetheobservedchangesinEEGpatternsandFOSexpressionwithnociception.

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Animals2022,12,2309 3of17

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Animals2022,12,2309 3of17 Inthisstudy,electroencephalographicreactionsinthecerebralcortexandchangesinFOSproteinexpressioninthespinaldorsalhornofsurgicallycastratedmalepigletsunderlightisofluraneanaesthesiaaredescribed.Thesemeasurementswereevaluatedasadditionalparametersofnociceptioninamultiparametricstudy.Thecorrespondingresultsforhaemo-dynamicandneurohumoralmeasurementsandtheevaluationoflimbmovements,whichwereinvestigatedinthesamepiglets,arepresentedinastudybySalleretal.[3].2.MaterialsandMethodsThestudywasperformedincompliancewiththeEUDirective2010/63/EUforanimalexperimentsandtheGermanAnimalWelfareAct[32].AllprocedureswereapprovedbytheEthicsCommitteeforAnimalExperimentsoftheGovernmentofUpperBavaria,Munich,Germany(ReferenceNumberROB-55.2-2532.Vet_02-19-11).2.1.AnimalsForty-ninehealthymalehybridGermanLandrace/GermanLargeWhite Pietrainpigletsfrom15littersagedthreetosevendayswereincludedinthisstudy.PregnantsowsweresourcedfromacommercialpigletproducerandtransferredtotheanimalhusbandryunitoftheClinicforSwine(CenterforClinicalVeterinaryMedicine,LMUMunich,Oberschleissheim,Germany).SowsandpigletswerehousedaccordingtotheGermanOrderfortheKeepingofProductiveAnimalsandtheEUDirective2010/63/EUforanimalexperiments.Usingacomputer-generatedrandomisedgroupassignment,animalsweredistributedintosixtreatmentgroups:fourgroupsreceivedeither4%procainehydrochloride(1),2%lidocainehydrochloride(2),0.5%bupivacainehydrochloride(3)or2%mepivacainehydrochloride(4)asalocalanaesthetic,onegroupreceivedaninjectionofsalinewithoutfurtheranalgesia(5)andonehandlinggroupunderwentonlysimulatedinterventions(6).Thestudywasdesignedasarandomised,double-blindstudy.2.2.ExperimentalProtocolAnaesthesiawasinducedwithaninitialconcentrationof5%isoﬂurane(IsoﬂuraneBaxtervet.,BaxterDeutschlandGmbH,Unterschleissheim,Germany)in3L/minoxygenviaafacemask.Pigletsbreathedspontaneouslyduringtheentiretrial.Whenthemusclesrelaxedandtherightingreﬂexwasnolongerelicited(noreactionwhentheanimalwasplacedinthesupineposition),theconcentrationofinspiratoryisoﬂuranewasreduced.Aftertheinductionofanaesthesia,ﬁve27-gaugedisposablestainlesssteelneedleelectrodeswereplacedsubcutaneouslyontheheadofthepigletasfollows:recordingelectrodeswereplacedsupraorbitallyintheareaofthefrontalboneonbothsidesofthehead,tworeferenceelectrodeswereplacedsidebysideinthemidlineoftheheadatthehighestpointoftheskull,andthegroundelectrodewasplacedintheneck.CorticalsignalswererecordedcontinuouslythroughouttheexperimentwithacommercialEEGmonitor(Narcotrend©,MTMonitortechnikGmbH&Co.,KG,BadBramstedt,Germany).EEGdatawererecordedfromtwobipolarchannelswithasamplingrateof128Hz.AfterplacingtheEEGelectrodes,thepigletwasturnedtothesupinepositiontoinsertcathetersintothecarotidarteryandthejugularveintomonitorbloodpressureandheartrateandwithdrawbloodsamples.Duringallnoxiousinterventions,theoccurrenceoflimbmovementswasalsorecorded(adetaileddescriptionoftheprocedureandresultshasbeenpublishedbySalleretal.[3]).Followingthemeasurementpreparations,inspiratoryisoﬂuranewasfurtherreducedto1.5%.Theappropriateconcentrationofisoﬂurane(presenceofnociceptivewithdrawalreﬂex)wasevaluatedineverypigletindividuallybyapplyingaPeanclamptopinchtheinterdigitalskinfoldofthehindlimb.Theclampwasclosedmaximallytotheﬁrstratchetforamaximumtimeof5soruntilmovementwaselicited,whicheverperiodwasshortest.Ananaestheticlevelallowingforaslightmovementreactionofthestimulatedlimb,provokingneitherinappropriatelyintensemovementreactionsnorawakeningthe

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Animals2022,12,2309 4of17

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Animals2022,12,2309 4of17 piglet,wasdeterminedtobeappropriate.Ifnomovementreactionoccurred,theinspiredisoﬂuraneconcentrationwasreducedin0.2%stepsandsubsequentlyadjusteduntilaslightlimbmovementoccurred.Incontrast,prolongedpaddlingand/ormovementoftheforelimbs,backorheadwereassessedasbeingtooexcessivefortheexperimentalsetup,andtheinspiredisoﬂuraneconcentrationwasincreasedinstepsof0.2%andequili-bratedfor3min.Thisapproachresultedinameanend-tidalisoﬂuraneconcentrationof1.69 0.3%forcastration.Aftertheadequatehypnoticstatewasreached,aninjectionofeitherlocalanaestheticsorsalinewasperformed,exceptinthehandlinggroup,whereinjectionsandsurgicalinterventionswereonlysimulated.ThetimelinefortheoverallexperimentispresentedbySalleretal.[3].Allpiglets,exceptforthehandlinggroup,receiveda0.5mLintratesticularanda0.5mLsubscrotalinjectionpertestis.Theappliedlocalanaestheticswere0.5%bupivacainehydrochloride(Bupivacain0.5%,JENAPHARM,MibeGmbHArzneimittel),2%lidocainehydrochloride(Xylocitin®2%,MibeGmbHArzneimittel,Brehna,Germany),2%mepiva-cainehydrochloride(Mepidor®20mg/mLsolutionforinjectioninhorses,RichterPharmaAG,Wels,Austria)and4%procainehydrochloride(4%procainehydrochloride-VMD,V.M.D.sa,Arendonk,Belgium).ThepHvaluesofthefourlocalanaestheticsrangedfrom3to6.5(pHvaluesaccordingtothemanufacturer:4%procainehydrochloride3–4.5,0.5%bupivacaine4–6,Mepidor®~5.4,and2%Xylocitin®5.5–6.5).Twocontrolgroupswereincludedinthestudy:onegroupthatreceivedasalineinjection(0.9%NaCl,castrationwithoutpainrelief)andtheshamgroupthatunderwentonlyasimulatedinjectionandcastration(handling).Anautomaticself-ﬁlling1mLsyringe(HSWECO-MATIC®,Henke-Sass,WolfGmbH,Tuttlingen,Germany)witha25Gcannula(0.5 16mm,B.BraunTravaCareGmbH,Hallbergmoos,Germany)wasusedfortheinjection.Thetestiswasﬁxedwiththethumbandindexﬁngerfortheintratesticularinjection.Subsequently,asmallskinfoldwasformedoverthetestis,whilethecannulawasretracteduntilthetipwaslocatedsubcutaneously,andthesubscrotalinjectionwasperformed.Thehandlinggroupwasmanipulatedandﬁxedinthesamemannerastheothergroups.Theautomaticsyringewaspushedonlyslightlyagainstthetesteswiththeneedlecappedtosimulatetheinjectionsinthisgroup.Afteranexposuretimeof20min,surgicalcastrationwasinitiated(exceptinthehandlinggroup).Inthecaseofcastration,theskinwasopenedwithascalpelbycreatingtwosagittalincisionsdowntotheviscerallayerofthevaginaltunic,andthetesteswereexposed.Afterastabilisationperiodoftwominutes,thetestesweregrasped,andthespermaticcordsweresuccessivelyseveredwithanemasculator.Thebluntbackofascalpelhandlewasmovedoverthescrotumtoimitatetheskinincisionintheanimalsofthehandlinggroup.Anaesthesiawasmaintainedforanother90minbeforeeachpigletwaseuthanisedwithanintravenousoverdoseofpentobarbital(Euthadorm500mg/mL,CPPharma,Burgdorf,Germany).FortheevaluationofEEGreactions,theEEGrawtraceswerevisuallyinspectedforartefactsorEEGburstsuppression(BSupp)patternsofﬂine.BecauseBSuppEEGcharacteristicsstronglydeviatefromtheEEGpatternspresentundergeneralanaesthesiawithoutBSupp,thedecisionwasmadetoexcludethesesegmentsfromfurtheranalysis.Allanimalsreceivedtheentiresetofnoxiousstimuliexceptforthehandlinggroup,inwhichonlyinterdigitalpinchesandsimulatedinterventionsweretested.Inthepigletsthatunderwentmorethanoneinterdigitalpinchfortheadjustmentofanaestheticdepth,thepinchthatwasincludedintheanalysiswastheoneatwhichtheanaestheticdepthwasevaluatedtobeappropriate.Eachbilateralinterventioninjection,skinincisionandcuttingofthespermaticcordwasperformedwithinafewsecondsonbothtestes.Theinjectionrequiredapproximately9stocompletepertestis,andskinincisionsanddissectionsofthespermaticcordrequired

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Animals2022,12,2309 5of17 approximately6seachtocomplete.TheEEGwasanalysedoveratimespanof90sfollowingthebilaterallyperformedstimulitofullyassesstheresponsetothenociceptivestimulus.Immediatelyaftereuthanasia,lumbarandsacralsegmentsofthespinalcordweredissectedfromthesurroundingtissueandremovedforFOSmeasurements.Usingthevertebraeaslandmarks,thespinalcordwascutintothreetransverselumbarsegments(L1,L2,andL3)andonesacral(S1-S3)spinalcordsegment,andﬁxedwith38%(w/w)neutral-bufferedformaldehydeforatleast48handembeddedinparafﬁn(LeicaASP300S,Leica,Wetzlar,Germany).Consecutivesections(2 m)wereobtainedfromthecranialsectionofeachparafﬁnblockandstainedwithhaematoxylin–eosin.FOSimmunohistochemistry(anti-c-FOSantibody,ab209794,Abcam,Cambridge,UK,dilutedinantibodydiluent1:100)wasperformedusingaLeicaBondRXmsystem.Brieﬂy,afterdeparafﬁnisation,pretreatmentwasperformedwithEpitopeRetrieval1(correspondingtocitratebufferpH6)for30min.Forprimaryantibodybindingdetectionandvisualisation,apolymerreﬁnedetectionkitwasusedwithoutapostprimaryantibodyandwith3,3’-diaminobenzidine(DAB)asthechromogen.AllslideswerescannedwithaLeicaAT2scanningsystem.Averticallinewasdrawnfromthecentralcanaltothelateralborderofthegreymattertostandardisetheareaforanalysis.Thespinaldorsalhornwasdeﬁnedasthegreymatterdorsaltothelinethatwasdrawn(Figure1B).ThestainingintensityandfrequencyofFOS-positiveneuronsinthespinaldorsalhornwereevaluatedsemiquantitatively(Figure1)byaGermanboard-certiﬁedpathologistwhowasblindedtothegroupsusingAperioImagescopeSoftwareversion12.4.0.7081.

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hornwereevaluatedsemiquantitatively(Figure1)byaGermanboard-certiﬁedpathologistwhowasblindedtothegroupsusingAperioImagescopeSoftwareversion12.4.0.7081. Figure1.SemiquantitativeEvaluationoftheFOSStainingIntensity.(A)ScorefortheFOSstainingintensityandfrequency.(B)Areaevaluatedinthespinalcord.(C)Examplesofdifferentscores.Theanalysismethodwasﬁrstestablishedinapreliminarystudy(datanotshown)todevelopanddeﬁneascoringapproachforformalin-ﬁxed,parafﬁn-embeddedandstainedtissuesections.Thestainingintensity(none,slight,moderate,orstrong)andthepercentageofpositiveneuronalnucleiinbothdorsalhornsofthespinalcordswerecalculated.Asemiquantitativescoreincludingthesetwoparameters(Figure1A)wasapplied.Fourdifferentsegments(L1,L2,L3,andS1-3)ineachanimalwereevaluated.Theresultsofthisevaluationclearlyshowedthehighestscoresintheanimalscastratedwithoutpainrelief.Thus,itwasdecidedtoproceedwiththismethodologicalapproach.

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Animals2022,12,2309 6of17 2.3.StatisticalAnalysis2.3.1.EEGThepowerspectraldensityoftheselectedEEGepisodeswascalculatedwiththeMATLABpwelchfunctionandafrequencyresolutionof0.25Hzforthepooleddatafromtheinterdigitalpinchstimulusand1Hzforthedatarecordedinresponsetootherstimuli.Becauseofthelargersamplesizeinthepooledgroupforthepinchstimulus,wedecidedtouseahigherfrequencyresolutiontobetterdescribetheresponse.Densityspectralarrayswerealsoconstructed,andtheresultsarepresentedasheatmapstodepictthetemporalevolutionofthespectralEEGfeatures.TheDSAwasderivedfrom5-sEEGepisodesoverlappingby4s.TheresultsarepresentedaschangesintheEEGpowerrelativetoaprestimulusbaselinedeﬁnedastheepisode50–40sbeforethestimulus.Thepoststimulusvaluewasdividedbytheprestimulusvalue.Thus,avalue<1indicatesahighervaluebeforethestimulus,whereasavalue>1indicatesanincreaseafterstimulation,andavalueof1indicatesnochangeafterthestimulus.Thisapproachhelpstotrackstimulus-inducedEEGchangesandcorrectfordifferentEEGamplitudesbetweentheanimals.FortheDSAplots,asignedranktestwasperformedforeachpixelbyassessingthepoweroftherespectivefrequencyattheobservedtimeversusthepowerofthefrequencyatbaseline.Pixelsthateitherhadap<0.05(pooledresponsetotheinterdigitalpinch)orp<0.1(singleexperimentalgroup,allotherstimuli)areshown.Hence,onlysigniﬁcantchangesthatoccurredinclustersarediscussed.Theclusterapproachwasusedbecauseofthelowprobabilityoftheconcentrationofspuriousfalsepositivesinacluster.Therefore,thestrategywasusedtodeﬁneaclustersize[33],i.e.,eithera4s 2Hzclusterforthepooledreactiontotheinterdigitalpinchstimulusora4s 4Hzclusterforthereactiontootherstimuli.Theareaunderthereceiveroperatingcurve(AUC)wascalculatedwith10k-foldbootstrapped95%conﬁdenceintervalstotrackthechangesintheEEGbandpowerovertime.Ifthe95%conﬁdenceintervalsdidnotcrossthe0.5line,thisresultwasconsideredtobesigniﬁcantaslongasitwasobservedinatleastattwoneighbouringtimepoints.Similarapproacheshavebeenusedpreviously[34,35].MATLABR2017a(TheMathWorks,Inc.,Natick,MA,UnitedStates)wasusedforthestatisticalanalysis.FortheAUCcalculation,additionalfunctionsfromthemeasuresoftheeffectsizetoolboxwereused[36].Differentsettingswereusedfortheinterdigitalpinchstimulusandtheothernoxiousstimuli,asamuchlargernumberofsampleswasavailablefortheinterdigitalpinchbypoolingdatafromallanimals,independentofthetreatmentgroups.BeeswarmplotsweregeneratedwiththeMATLABplotSpreadfunction.2.3.2.FOSProteinFourtissuesectionsperanimalwereincludedintheanalysis:onesectioneachfromspinalcordsegmentsL1,L2,L3andS1-3.Themedianstainingintensityandfrequencyofthedark-brownDABprecipitateinthefourspinalcordsegmentswerecalculatedforeveryanimal,andaKruskal–WallistestwasappliedtoidentifysigniﬁcantdifferencesbetweentheNaClgroupandtheotherexperimentalgroups.Figure1showsthescoringschemeappliedfortheFOSanalysis.3.Results3.1.DemographicsFortheEEGanalysis,196datasets(fourperanimal)from49pigletsaged5.3 1.1daysandweighing2.14kg 0.45kgwereacquired.SomeanimalsexhibitedtimespanswithBSuppinmeasurementperiods;therefore,30datasetswerediscarded(27duetoBSupppresenceand3duetoartefacts),and166datasetswereincludedintheanalysis.Theinter-digitalpinchwasperformedunderisoﬂuraneanaesthesiaintheabsenceofanyanalgesic.Itwasappliedtoallanimalsusinganidenticaltechnique.Therefore,theanimalsfromallexperimentalgroupswerecombinedtoevaluatetheEEGchangesduetotheinterdigitalpinch(n=44datasets).Forthesereasons,varyingnumbersofdatasetswereanalysedforthesixexperimentalgroups(Table1).

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Animals2022,12,2309 7of17 Table1.Overview:NumberofDatasetsUsedintheEEGAnalysis. ExperimentalGroup NoxiousStimulus InterdigitalPinchInjectionIncisionCuttingoftheSpermaticCord NaCl(n=8animals) n=44datasets(pooleddata)n=7datasetsn=6datasetsn=6datasetsLidocaine(n=9animals) n=7datasetsn=8datasetsn=7datasetsBupivacaine(n=9animals) n=9datasetsn=9datasetsn=9datasetsMepivacaine(n=6animals) n=5datasetsn=5datasetsn=5datasetsProcaine(n=8animals) n=6datasetsn=5datasetsn=5datasetsHandling(n=9animals) n=7datasetsn=8datasetsn=8datasets Totaln=49animals Totaln=166datasets FortheFOSanalysis,tissuesectionsfrom42animalswereevaluated.Duetosamplingandtrimminginaccuracies,theindividualspinalcordsectionsfromsevenanimalscouldnotbeevaluated.Therefore,thosesevenanimalswereexcludedfromtheﬁnalanalysis.Theanalysedanimalswere5.5 1.0daysofageandweighed2.13kg 0.45kg(Table2).Table2.Overview:NumberofAnimalsfortheFOSAnalysis. FOSproteinAnalysisNaClLidocaineBupivacaineMepivacaineProcaineHandling (n=42animals)n=7n=7n=8n=7n=6n=7 3.2.InterdigitalPinchForthepooleddata(n=44),asigniﬁcantincreaseinfrequenciesof~10–14Hzwasobservedimmediatelyafterthestimuluswasapplied,alongwithasigniﬁcantdecreaseinthe~0.5–8Hzbandpower(deltaandthetarange,‘early’response).Thedecreaseinpowerlasteduntilapproximately35safterthebeginningofstimulation(t=0).Afterthisdecreaseinpower,anincreaseinpoweratfrequencies>10Hzwasobservedstartingafterapproximately35suntilapproximately90saftert=0(‘late‘response).Figure2presentstheDSAofthechangeinpowerrelativetothebaselineandhighlightstheearlyandlatecomponentsobservedfollowingtheinterdigitalpinch.

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ate‘response).Figure2presentstheDSAofthechangeinpowerrelativetothebaselineandhighlightstheearlyandlatecomponentsobservedfollowingtheinterdigitalpinch. Figure2.DSAofthePooledMedianResponse(n=44)toInterdigitalPinchStimulationover90s.Areaswithboldcoloursindicatethefrequencyandtimeregionswithsigniﬁcantdifferencesbetweenpre-andpoststimulusconditions.Thecolourbarindicatesthechangeinthepoststimuluspowercomparedwiththeprestimulusbaseline.Thestimulusstartedatt=0.Brieﬂy,theinterdigitalpinchleadstoan‘early‘reductioninpowerintheslowfrequenciesbelow10Hzanda‘late‘activationofhigherfrequenciesabove10Hzstartingapproximately35safterthestimulation.3.3.InjectionsThereactionstotheinjectionstimuliwerequiteheterogeneousamongthesubstances.FortheNaCl,bupivacaineandprocainegroups,asigniﬁcantreactionwasobservedasanactivationoffrequenciesof~10–15Hzatapproximately30–40safterthestimulus.Inparticular,intheprocainegroup,asigniﬁcantongoingdepressionof~10–15Hzoscillationsappearedstartingapproximately60safterthestimulus.Forthemepivacaineandprocainegroups,areductioninpowerinthelowerfrequenciesimmediatelyaftertheinjectionstimuluswasdetected.Lidocaineandbupivacaineproducedshort,signiﬁcantdecreasesatapproximately80s,atfrequenciesof~10–15Hz.

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Animals2022,12,2309 8of17 SupplementalFigureS1presentstheDSAsofthechangesinpowercomparedwithprestimulusconditionsforthe90speriodaftertheintratesticularandsubscrotalinjections(FigureS1).3.4.SkinIncisionAbiphasic,earlyandlateresponsetothescrotalskinincisionwasobservedintheNaClgroupwithan‘early’(~10s)decreaseof~5–15Hzpowerfollowedbya’late’activationof~20–30Hzpower,startingatapproximately25s.Forallothergroups,asignificant,stimulus-inducedchangewasnotobserved.Figure3presentstheDSAsofthechangeinpowerwhencomparedwiththeprestimulusconditionsforthe90speriodaftertheincision. Figure3.DSAsoftheChangesinEEGsaftertheScrotalSkinIncision.TheDSAspresentchangesinEEGpowerover90sfollowingtheincisionstimulusinthefollowingexperimentalgroups:NaCl(A),lidocaine(B),bupivacaine(C),mepivacaine(D),procaine(E),andnoincisionandhandlingonly(F).Theincisionstartedatt=0.AreasshowninboldcoloursindicatefrequencyandtimeregionswithasigniﬁcantdifferencefromtheprestimulusEEG.(A)NaClcausedan‘early‘and‘late‘responsetotheincisionstimulus.Theearlyresponsewasanattenuationinthe5–15Hzrangewithintheﬁrst20safterstimulation,andthelateresponsewasanactivationinthe20–30Hzpowerrangeatapproximately30–40safterthestimulation.(B–F)SigniﬁcantchangesintheEEGpowerwerenotobservedinthelocalanaestheticandhandlinggroupsfollowingthestimulus,exceptforahigh-frequencyactivationatapproximately30Hzandapproximately60safterstimulationinthebupivacainegroup(C).

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ndhandlinggroupsfollowingthestimulus,exceptforahigh-frequencyactivationatapproximately30Hzandapproximately60safterstimulationinthebupivacainegroup(C). Animals2022,12,2309 9of17 3.5.CuttingoftheSpermaticCordAnalogoustotheincisionandtheinterdigitalpinch,abiphasicresponsetospermaticcorddissectionwasdetectedintheNaClgroup,withanearly(~10s)decreasein~5–15Hzpowerfollowedbyalateactivationof~20–30Hzpowerstartingatapproximately20s.Thisbiphasicresponsewasnotobservedinanyoftheothergroups.Forthelidocainegroup,anincreasein~10Hzpowerwasobservedapproximately40safternoxiousstimulation.Forallothergroups,nospeciﬁcreactiontothestimuluswasdetected.Figure4presentstheDSAofthechangeinpowerforthe90speriodafterspermaticcorddissection,comparedwiththeprestimulusconditions. Figure4.DSAsoftheEEGChangesafterSpermaticCordDissection.TheDSAspresentchangesinEEGpowerover90sfollowingspermaticcorddissectioninthefollowingexperimentalgroups:NaCl(A),lidocaine(B),bupivacaine(C),mepivacaine(D),procaine(E),andnoincisionorhandlingonly(F).Stimulusstartedatt=0.(A)IntheNaClgroup,an‘early‘and‘late‘responsetothedissectionstimuluswasobserved.Theearlyresponsewasanattenuationinthe~5–15Hzrangewithintheﬁrst15safterstimulation,andthelateresponsewasanactivationof~20–30Hzatapproximately20and30–40safterstimulation.(B–F)NosigniﬁcantchangesintheEEGpowerwereobservedinthelocalanaestheticandhandlinggroupsfollowingthestimulus,exceptforanactivationof~10Hzpoweratapproximately40safterstimulationinthelidocainegroup(B)andalatedecreasein~15Hzpoweratapproximately80sinthebupivacainegroup(C).

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Animals2022,12,2309 10of17 3.6.EvaluationoftheEarlyandLateResponsesTheNaClgroupshowedasimilarreactionpatternfollowingskinincisionandcuttingofthespermaticcord,namely,anearlydecreaseinlow-frequencywavesfollowedbyanincreaseinhigh-frequencywaves(lateresponse).ThedetectionperiodsofalllocalanaestheticgroupsandthehandlinggroupwerecomparedwiththoseoftheNaClcontrol.Figure5presentsbeeswarmplotsofthestatisticalanalysesoftheearly(5–10safterstimulus)decreaseinthefrequencybandsof5–15Hzandthelate(30–40safterstimulus)increaseinthefrequencybandsof20–30Hz.Thereactionsofthedifferentsubstancegroupsandthehandlinggrouptoskinincisions(Figure5A,B)andspermaticcorddissections(Figure5C,D)werecomparedwiththeNaClgroup.

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actionsofthedifferentsubstancegroupsandthehandlinggrouptoskinincisions(Figure5A,B)andspermaticcorddissections(Figure5C,D)werecomparedwiththeNaClgroup. Figure5.Comparisonof‘early‘and‘late‘reactionsfollowingskinincision(A,B)andspermaticcorddissection(C,D),comparedwiththeNaClgroup.(A)Forskinincisions,asigniﬁcant(*)orpossiblyrelevant(AUC>0.7)effectwasobservedfortheearlyresponseinallgroups,exceptfortheprocainegroup.The5–15HzpowerdecreasedbyasmalleramountasareactiontothestimuluswhencomparedwiththechangeintheNaClgroup.(B)Regardingthelateresponse,allgroupsshowedaweakeractivationof20–30HzpowerasaresultofthestimulationcomparedwiththatoftheNaClgroup.(C)Asanearlyreactionfollowingspermaticcorddissection,allgroupsexhibitedaweaker5–15HzresponsethantheNaClgroup.(D)Signiﬁcantdifferencesinthelate20–30Hzpowerresponseswerenotobservedafterspermaticcorddissection.3.7.FOSProteinSigniﬁcantlylowerexpressionoftheFOSproteinwasobservedintheanalysedlumbar(L1,L2,L3)andsacral(S1-3)segmentsofthespinaldorsalhorninthehandlinggroupandinalllocalanaestheticgroups,exceptthebupivacainegroup,whencomparedwiththeNaClgroup.Allanimals,includingthehandlinggroup,displayedatleastslightstainingin<25%oftheneuronsthatresultedinaminimumscoreofone.Figure6displaysthecorrespondingscatterplotaswellasimagesoftheimmunohistochemicalstaining.Additionally,FOSproteinexpressionwasevaluatedonlyinanimalsthatshowednoBSupppatternsthroughoutthemeasuredEEGepochs(FigureS2).

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Animals2022,12,2309 11of17

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Animals2022,12,2309 11of17 Figure6.ScatterplotoftheFOSstainingscore(A)andrepresentativeimagesofFOSproteinexpression(B–E).(A)Signiﬁcantly(AUC95%CIexcept0.5)lessFOSproteinexpressionwasobservedinthehandlinggroupandinalllocalanaesthesiagroups,exceptforthebupivacainegroup,thanintheNaClgroup.(*)Asigniﬁcanteffect(p<0.05).(B–E)RepresentativeimagesofFOSproteinexpressioninthedorsalhornofthespinalcordinanimalsfromtheNaCl(B),lidocaine(C),bupivacaine(D),andhandling(E)groups(barsindicate30 m).4.DiscussionTheinitialanalysisofthechangesinEEGpatternsfollowingtheinterdigitalskinfoldpinch,whichinducedanociceptivewithdrawalreﬂex,showedastrongbiphasicresponse.Asthisstimuluswasappliedtoallanimalsinanidenticalmannerwithoutanalgesia,thedatawerepooled.Thisapproachyieldedalargenumberofsamples.ThebiphasicEEGresponseconsistedofan‘early’anda‘late’component.SimilarEEGreactionswererecordedfollowingthescrotalskinincisionanddissectionofthespermaticcordwithoutlocalanaesthetics.Theapplicationoflocalanaestheticsattenuatedcorticalreactions.Additionally,FOSproteinexpressioninthedorsalhornofthespinalcordwasreducedbymostlocalanaesthetics.Usinganinterdigitalpinch,whichwasreportedtobeastrongnoxiousstimulusinnewbornsandjuvenilepigs[37,38],theadequateanaestheticlevelforeachanimalwasdeterminedindividually.Thepresenceofanociceptivewithdrawalreﬂexinducedbyinterdigitalpinchingisawell-acceptedindicatorofnociceptionduringanaesthesiainvariousspeciesandinpigs[39–41].BSupp,apatternofwaxingandwaningamplitudes,mayreﬂectastateofexces-sivelydeepanaesthesia,butitmayalsooccuratlowanaestheticdosesinthedevelopingpig[42,43].Inthepresentstudy,somepigletsshowedtimespanswithBSupp,andthustheywereexcludedfromtheanalysis.Inapreviousstudyofjuvenilepigs,individualanimalsalsoexhibitedBSuppatisoﬂuraneconcentrationsofsub-MAClevels[44].Ingeneral,isoﬂuranedepressesthedose-relatedcorticalelectricalactivityinjuvenilepigs[42].However,theoccurrenceofmovementreactionstonoxiousstimulationinjuvenilepigsandratswithBSupphasbeenreported[37,45,46].4.1.EEGReactionPigletcastrationincludestwodistinctpainfulaspects,incisionofthescrotalskinandpullingandcuttingofthespermaticcords,whichissuggestedtobethemostpainfulcomponentofcastration[3,47].IntheEEGmeasurements,bothnoxiousstimuliwereanalysedseparately.ThecorticalreactionsintheNaClgroupwereexpectedtoreﬂecttheunalteredandmostintensenoxiousstimulusinthisexperimentbecausenoanalgesiawasapplied.Forthisgroup,abiphasicresponsetoboththeskinincisionandcuttingofthespermaticcordswasobservedthatwassimilarinshapetotheinterdigitalpinchresponse.TheEEGreactionwasalteredorstronglyattenuatedinthelocalanaestheticandhandlinggroups.Afterapriorapplicationofeitherlidocaine,mepivacaineorprocaine,theincisionofthescrotalskinevokednosigniﬁcantreactionsinthecerebralcortex,oratleastastronglyattenuatedreaction,inthebupivacainegroup.Followingthecuttingofthespermaticcords,ashortbutsigniﬁcantactivationwasobservedinthelidocainegroup,but

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Animals2022,12,2309 12of17 nosigniﬁcantchangesweredetectedintheotherlocalanaestheticgroups.ThecomparisonoftheintensechangesduringtheearlyandlateresponseobservedintheNaClgroupwiththatinthegroupsthatreceivedlocalanaestheticandthehandlinggroupalsosuggestsweakerresponsesinthelocalanaestheticgroupsandinthehandlinggroup.TheEEGreactiontonoxiousstimulationundergeneralanaesthesiamaybemani-fold[6].Inpatientsundergeneralanaesthesia,noxiousstimulationmaydecreaseEEGpowerinthealphaband[48,49]orincreasethepowerinthedeltaband[50]orbetaband[51].VariousEEGreactionstonoxiousstimulationthatmaydependonthespecies,age,thetypeanddoseofgeneralanaestheticused,thetypeandintensityofstimulationandtheamountofanalgesichavealsobeenreportedforinterventionsinanimals[8,11,12,44,52,53].TheobservedEEGreactiontotheinterdigitalpinchwithanearlydecreaseinslowoscillatoryactivityfollowedbyanincreaseinoscillatoryactivityabove10Hzmostlikelyindicatesachangetowardsarousal.Theadditionalstrongincreaseinpowerinthe10–15Hzrangeduringtheﬁrstsecondsafterthestimulusbeganmaybeattributedtotheclampthatwasstillattachedtotheskinfold.EEGarousalindicatesashiftinEEGactivityfromthelowertothehigherfrequencyrange.Thesehigherfrequenciesarisefromadesynchronisationofneuronalactivityandareusuallyassociatedwithhighercorticalfunction[54].ThearousalreactionmayberegardedasachangeintheEEGpatterninthedirectionoftheconsciousEEGpattern[17].Itisnotaspeciﬁcindicatorofnociceptiveinputbutrepresentsacommonreactiontonoxiousstimulationinhumansandhasalsobeendescribedinanimals[9,14,18].Hagaetal.[22]didnotobservesigniﬁcantEEGchangesindevelopingpigsfollowinginnocuousnoxiousmechanicalstimulationunderisoﬂuraneanaesthesia.Anunderlyingbiphasicresponsepatternasobservedinthepresentstudymayhavemaskedtheﬁndings.Otherﬁndingsseemtobesimilartothepresentresults.EEGdatarecordedinadultgoatsandaveragedover1minshowedthatthegoatsreactedtoaclamponthedewclawunderisoﬂuraneanaesthesiawithdecreasesintheprocessedparameterstotal,delta,thetaandalphapower,whilethebetapowerwasunchanged[10].Nevertheless,theresultsmaynotbedirectlycomparablewiththoseofthepresentstudyduetodifferencesinseveralparameters,e.g.,species,age,EEGsegmentlengthusedforanalysisandtheuseofprocessedEEGparameters.EEGmeasureswerealsousedtoexaminesurgicalnoxiousstimulationinducedbytaildockinginpiglets.AcomparisonofEEGresponsesinpigletsofeither2or20daysofageshowedaneffectofageontheEEGreaction[19].YoungpigletsshowedlittleEEGresponsetotaildocking,whereasolderpigletsreactedwithsigniﬁcantchangesinmedianfrequencyandtotalpower.Johnsonetal.alsodetecteddifferentEEGresponsesinpigletsbetween1and15daysofage[21].ThevaryingEEGreactionsofpigletsintheﬁrst20daysoflifeimplythatmajorchangesoccurintheEEGresponsesofpigletstonoxiousstimulation,particularlyduringtheﬁrstweeksoflife.BothstudiesdocumentedchangesinprocessedEEGparametersfollowingtaildockingunderhalothaneanaesthesia,butdetailedEEGdataaremissing.DifferentnoxiousstimulicausedifferingEEGchangesaswell[12].Therefore,acomparisonwiththepresentresultsisonlypossibleunderrestrictions.StudiesofEEGresponsestosurgicalcastrationinpigletsexist,althoughnoneofthesestudieswereperformedunderisofluraneanaesthesia.However,theanaestheticagentusediscrucialbecausedifferentanaestheticagentsinducedifferentEEGpatterns[34,53,55,56],andthechoiceofanaestheticagentmaypartiallyaccountfordifferencesinresultsbetweenEEGpatternsinnociceptivestudies[7].InitialfindingsfromWaldmannetal.[18]onthecastrationofpigletsalsorevealEEGarousalreactionsintheEEGsofindividualpigletsfollowingcastrationunderbarbiturateanaesthesia.HagaandRanheim[17]observeddecreasesindelta,theta,andalphabandsandtotalEEGpowerfollowingcastrationunderhalothaneanaesthesiawithoutadditionalanalgesiainapproximatelythree-week-oldpiglets.PigletsthatreceivedlidocaineeitherintrafunicularlyorintratesticularlyshowedalesspronouncedEEGresponse[17].Thepresentedresultstendtobesimilar,asreactionsinthecerebralcortexweredecreasedoratleastalteredafterlocalanaesthesiawasapplied.BecauseoftheuseofEEG

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Animals2022,12,2309 13of17 bandpowerandthelackofdetailedinformationregardingchangesintheEEGspectruminthesestudies,comparingthedetailedEEGresponsesisnotpossible.TheinjectionoflocalanaestheticsyieldedtheexpectedheterogeneousEEGreactions.ThedataindicatethatintratesticularandsubscrotalinjectionsprovokesigniﬁcantchangesinEEGwaves,irrespectiveofwhichﬂuidisused.Thereactionsdifferedinallgroupsinregardstothebiphasicreactionpatternobservedfollowingtheinterdigitalpinch,ornoxiousstimulationbyskinincisionanddissectionofthespermaticcordwhennolocalanaestheticswerecoapplied.ThevaryingpharmacologicalpropertiesofthelocalanaestheticsmaycausedifferentstimulithatarenotuniformandmightexplainthevariouspostinjectionEEGpatterns.Nonetheless,thedatasetsfromtheinjectionsareconsideredvaluableandarepresentedassupplementaldata(FigureS1).ForthepresentationoftheEEGresults,thepowerspectrumusedforthegenerationoftheDSAplotsfrom5sEEGepisodeswitha1soverlapwascalculatedwiththegoalofnotmissinganyEEGreactionswithashortduration.Althoughthep<0.1thresholdisunusual,itmayhelpusbetteridentifythepossibleresponse;however,duetothelimitedsamplesize,theﬁndingsfromthedifferentgroupsarepreliminaryresults.Thestudyrefrainsfromdiscussingscatteredpixelsbecauseoftheincreasedriskofdiscussingfalsepositivesduetomultiplecomparisons,whichiswhytheclusterapproachwasused.Werefrainedfromfocusingonchangesinpredeﬁnedfrequencybandsorinparametersusinginformationfromtheentirespectrum.First,thedivisionoftheEEGpowerspectrumofanimalsintothefrequencybandsalpha,beta,deltaandthetamaybequitearbitrary[7],andvariabilityhasbeenobservedinthespeciﬁcfrequencyrangethatdeﬁneseachband[57].Second,processedparameters,suchasthemedianfrequencyorwidefrequencybands(e.g.,thebetarangefrom~12–30Hz),mightmaskeffects.Forinstance,anincreaseinpowerfrom12–16Hzandasimultaneousdecreaseinpowerfrom20–25Hzwouldnothaveaneffectonthebetaband.Ingeneral,differencesintheanaestheticagentused,noxiousstimuli,species,ageoftheanimals,evaluatedparameters,andtimescalespreventacomparisonofstudies.ThisstudypresentstheunprocessedspectralresponseintheEEG,whichmayfacilitatethetranslationoftheﬁndingstotheresultsfromotherexperiments.TheuseofDSAplotsandthedecisiontorefrainfromusingprocessedEEGparametersallowedustoobservechronologicalchangesinEEGactivitypatternsovertheentirefrequencyrange.ThisapproachdescribestheEEGreactionsinamoredetailedmannerthanusingprocessedEEGparametersorbandpowersderivedfromthespectrum.4.2.FOSProteinTheexpressionoftheimmediateearlygenec-FOSanditsproteinproductFOSinneuronsofthespinalcordisawell-establishedmarkertoquantifyneuralactivityinresponsetonoxiousstimulation[25,26].Noxiousperipheralstimulationevokesarapidchangeingeneexpressionofthepostsynapticneuronsinthedorsalhornofthespinalcord[24].ThemRNAoftheimmediateearlygenec-FOSisdetectedwithinfiveminutesfollowingphysicalstress,suchasinjury[58].Thec-FOSgeneencodesthenuclearproteinFOS,whichisdetectedinthesameneuronswithaproteinpeakobservedatapproximately90–120minaftertheinductionofgeneexpression[23,25,59].Afternoxiousperipheralstimulation,immunohistochemicalFOS-positiveneuronsarepredominantlyobservedinlaminaeIandIIofthedorsalhorn,alongwithsomelabellinginlaminaeIII-VI[26,27].AsemiquantitativeapproachwasusedtoevaluateFOSexpressioninthespinaldorsalhorn,whichincludeslaminaeI–VI.Thisapproachhasnotbeenvalidatedforuseasastand-aloneread-outofneuronalstimulationinthespinalcord.However,incombinationwiththeEEGresults,itservesasanadditionalparameter,whichcanbeusedtofurtherevaluatethenociceptiveresponse.Themajorityofafferentneuronssupplyingtheporcinetestisandadjacentstructuresarelocatedindorsalrootgangliaoflumbarsegmentsonetothreeandsacralsegmentsonetothree[60–62].Therefore,thesespinalcordsegmentswereincludedintheanalysis.SigniﬁcantlylowerlevelsofFOSexpressionweredetectedinthenucleiofspinalneurons

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Animals2022,12,2309 14of17 inthepigletsthatwerecastratedafterapplicationofalocalanaesthetic,exceptforthebupivacainegroup,andinanimalsthatwereonlyhandled,comparedwithanimalsun-dergoingcastrationwithoutanalgesia.FOSexpressionwasadditionallyanalysedinonlytheanimalsthatdidnotshowBSuppthroughouttheexperimenttoevaluateapossibleeffectoftheanaestheticlevel(i.e.,BSupp).Theresultsweresimilarandarepresentedinthesupportinginformation(TableS2).TheFOSresultssupportdatafromastudybyNyborgetal.,whoreportedalowernumberofFOS-positiveneuronsinthespinaldorsalhorninpigletsundergoingcastrationwithlocalanaesthesiathaninpigletsundergoingsurgerywithoutanalgesia[29].FOSexpressioninthedorsalhornofpigletswasalsoreducedaftercastrationunderCO2anaesthesia[28].Additionally,localanaesthesiawaseffectiveatreducingthenumberofFOS-positiveneuronsinjuvenilepigsundergoinglaparotomywhencomparedwithanimalsundergoingsurgerywithoutanaesthesia[27].Inthepresentexperiment,allanimalsshowedatleastslightstaininginsingleneu-rons.ThisﬁndingmayresultfromthefactthatFOSexpressioninthisexperimentrep-resentsthecumulativeresponsetonoxiousstimulationoccurringinthetimeperiodof~90–120minbeforeharvestingthespinalcordandtransferredvianeuronsinL1,L2,L3andS1-3.Allanimalsexperiencedatleasttwointerdigitalpinches,thesecondofwhichwasappliedfourminutesfollowingspermaticcorddissectionandthereforeoccurredintherelevanttimeframe.Duetothesimilarinnervationoftheinterdigitalareaofthehindclaw,theinterdigitalpinchesverylikelycontributedtoFOSexpressioninthepresentstudy.Moreover,changesinheartrateandmeanarterialbloodpressureinthehandlinggroupwereobservedfollowinginjection,skinincisionandspermaticcorddissection[3].Obviously,thetouchingandﬁxationofthetestesalsoledtoadversereactions,whichpossiblyalsocontributedtothebaselineFOSexpression.DespitebaselineFOSexpression,signiﬁcantdifferencesinFOSexpressionwereevidentbetweentheanimalstreatedwithoutanalgesiaandanimalstreatedwithlocalanaesthesiaoronly-handledanimals.Therefore,weassumedthatFOSexpressionmaybeusedasanindicatorofnociceptionduringsuck-lingpigletcastration.However,furtherstudieswithlargeranimalnumberswouldbebeneﬁcialtoconﬁrmthisﬁnding.OnelimitationofthestudyisthatasamplesizecalculationfortheoverallstudywasnotperformedbasedonEEGeffectsorFOSexpression.Haga,TevikandMoerch[22]foundbloodpressuretobeamoresensitiveindicatorofnociceptioninjuvenilepigsthanelectroen-cephalographicparameters.Therefore,thesamplesizeestimationwasspecificallybasedonthecorrespondinghaemodynamicparameters.Hence,thepowerofthepresentstudymaybetoolowtodrawdefiniteconclusionsbasedonEEGandFOSmeasurementsasstand-aloneparametersregardingtheefficacyofthefourlocalanaestheticsforpigletcastrationortocom-parethemwitheachother.Furtherstudieswithalargersamplesizeareneededtodescribetheeffectsonthelocalanaestheticgroupsinmoredetailandtocomparethegroups.5.ConclusionsTheEEGreactionsof3-to7-day-oldpigletstoaninnocuousinterdigitalpinchandtosurgicalcastrationunderlightisofluraneanaesthesiaweretracked.BoththenoxiousstimulationcausedbyaninterdigitalpinchandbycastrationwithoutpainreliefwerevisibleintheEEGasabiphasic‘early’and’late’response.ThisEEGreactionpatternwasattenuatedintheanimalsthatreceivedlocalanaesthesiaandinanimalsofthehandlinggroup.WeareconfidentinouridentificationofthenociceptiveresponseintheEEGbecauseofthe(i)repetitivenatureofthesignalsobservedfollowinginterdigitalpinchingandsurgicalinterventionswithoutanalgesia,(ii)theattenuatedresponsetosurgicalinterventionwithlocalanaesthetics,and(iii)thereducedFOSexpressioninanimalstreatedwithlocalanaesthetics.Itcanbeassumedthattheuseoflocalanaestheticsinpigletsundergoingsurgicalcastrationunderisoﬂuraneanaesthesiaverylikelyattenuatesnociceptivetransmissionunderstandardisedlaboratoryconditions.TheEEGandFOSresultsareconsistentwiththesigniﬁcantlyreducedheartrateandbloodpressureandthedecreasedmovementreactionsthatweredetectedinthe

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Animals2022,12,2309 15of17 sameanimals[3].TheresultsoftheEEGandFOSmeasurementsincombinationwiththecorrespondingresultsforthehaemodynamicparametersandtheevaluationoflimbmovements[3]providevaluableinformationwithwhichtoformanoverallunderstandingofthenociceptiveresponseofpigletstocastrationwithorwithoutlocalanaesthetics.Additionally,theresultsfromtheEEGandFOSanalysesprovidestrongpilotdataforfurtherstudiesregardingEEGnociceptioninpiglets.Toourknowledge,thedetailedEEGreactionsofpigletstoanoxiousinterdigitalpinchandsurgicalcastration,aswellasadescriptionofhowlocalanaestheticsmayalterthispattern,havenotbeenpresentedtodate.Furtherinvestigationsarerequiredtodeterminewhetherorhowtheresultsrelatetopainperceptioninconsciouspigletsandunderﬁeldstudyconditions.SupplementaryMaterials:Thefollowingsupportinginformationcanbedownloadedathttps://www.mdpi.com/article/10.3390/ani12182309/s1,SupplementaryMaterial1:FigureS1:EEGChangesafterIntratesticularandSubscrotalInjections;Supplementarymaterial2:FOSProteinAnalysisinOnlyAnimalswithoutBSupp;FigureS2:ScatterPlotoftheFOSStainingScore(BasedonAnimalswithoutBSupp);andTableS2:NumberofAnimalsIncludedintheFOSProteinAnalysis(BasedonAnimalswithoutBSupp).AuthorContributions:Conceptualisation,S.Z.andC.B.;Datacuration,J.R.andM.K.;Formalanalysis,M.K.;Fundingacquisition,S.Z.andC.B.;Investigation,J.R.,J.W.,A.M.S.,J.F.,S.S.,P.D.,N.A.,T.G.,A.G.andR.M.;Methodology,J.R.,J.W.,A.M.S.,J.F.,S.S.,P.D.,N.A.,T.G.andK.S.;Projectadministration,S.Z.andC.B.;Resources,M.R.,G.S.andC.B.;Supervision,S.B.,M.H.E.,M.R.,S.Z.,G.S.andC.B.;Validation,J.R.,M.K.andK.S.;Visualisation,J.R.,M.K.andK.S.;Writing—originaldraft,J.R.andM.K.;Writing—review&editing,J.W.,A.M.S.,J.F.,S.S.,P.D.,N.A.,T.G.,A.G.,R.M.,S.B.,M.H.E.,M.R.,S.Z.,G.S.,K.S.andC.B.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:ThisresearchwasfundedbytheGermanFederalMinistryofFoodandAgriculture(BMEL,https://www.bmel.de)basedonadecisionoftheParliamentoftheFederalRepublicofGermany,grantedbytheFederalOfﬁceforAgricultureandFood(BLE,https://www.ble.de,grantnumber:2817HS016,2817HS013).InstitutionalReviewBoardStatement:ThestudywasperformedincompliancewiththeEUDirec-tive2010/63/EUforanimalexperimentsandtheGermanAnimalWelfareAct[32].AllprocedureswereapprovedbytheEthicsCommitteeforAnimalExperimentsoftheGovernmentofUpperBavaria,Munich,Germany(ReferenceNumberROB-55.2-2532.Vet_02-19-11).InformedConsentStatement:Notapplicable.DataAvailabilityStatement:Notapplicable.Acknowledgments:TheauthorswouldliketothankStephanKratzerandSebastianBerger,whosupportedusinestablishingtheelectroencephalographicmeasurements;EvaPrötzl,whohelpedtoprovidetheMATLABcodefortheclusteranalysis;MarionMielkeandOlgaSeelbach,whoperformedtheparafﬁnembeddingandcutting,aswellastheimmunohistologicalstaining;andJohannaBrandl,whoprovidedsigniﬁcantsupportinthepreparationofthispaper.ConﬂictsofInterest:Theauthorshavenoconﬂictsofinteresttodeclare.References1.Hansson,M.;Lundeheim,N.;Nyman,G.;Johansson,G.Effectoflocalanaesthesiaand/oranalgesiaonpainresponsesinducedbypigletcastration.ActaVet.Scand.2011,53,34.[CrossRef]2.Kluivers,M.;Hopster,H.;Spoolder,H.A.M.CastrationunderAnaesthesiaand/orAnalgesiainCommercialPigProduction;AnimalSciencesGroup:Lelystad,TheNetherlands,2007.3.Saller,A.M.;Werner,J.;Reiser,J.;Senf,S.;Deffner,P.;Abendschön,N.;Weiß,C.;Fischer,J.;Schörwerth,A.;Miller,R.;etal.Localanesthesiainpigletsundergoingcastration-Acomparativestudytoinvestigatetheanalgesiceffectsoffourlocalanestheticsonthebasisofacutephysiologicalresponsesandlimbmovements.PLoSONE2020,15,e0236742.[CrossRef][PubMed]4.Rose,J.;Arlinghaus,R.;Cooke,S.;Diggles,D.B.K.;Sawynok,W.;Stevens,D.;Wynne,C.D.L.Canﬁshreallyfeelpain?FishFish.2013,15,97–133.[CrossRef]5.Mathews,K.;Kronen,P.W.;Lascelles,D.;Nolan,A.;Robertson,S.;Steagall,P.V.;Wright,B.;Yamashita,K.Guidelinesforrecognition,assessmentandtreatmentofpain:WSAVAGlobalPainCouncilmembersandco-authorsofthisdocument.J.SmallAnim.Pract.2014,55,E10–E68.[CrossRef][PubMed]

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6.García,P.S.;Kreuzer,M.;Hight,D.;Sleigh,J.W.Effectsofnoxiousstimulationontheelectroencephalogramduringgeneralanaesthesia:Anarrativereviewandapproachtoanalgesictitration.Br.J.Anaesth.2021,126,445–457.[CrossRef][PubMed]7.Murrell,J.C.;Johnson,C.B.Neurophysiologicaltechniquestoassesspaininanimals.J.Vet.Pharmacol.Ther.2006,29,325–335.[CrossRef][PubMed]8.Otto,K.A.Effectsofaveragingdataseriesontheelectroencephalographicresponsetonoxiousvisceralstimulationinisoﬂurane-anaesthetizeddogs.Res.Vet.Sci.2007,83,385–393.[CrossRef][PubMed]9.Otto,K.A.;Mally,P.NoxiousstimulationduringorthopaedicsurgeryresultsinEEG‘arousal’or‘paradoxicalarousal’reactioninisoﬂurane-anaesthetisedsheep.Res.Vet.Sci.2003,75,103–112.[CrossRef]10.Antognini,J.F.;Carstens,E.Isoﬂuranebluntselectroencephalographicandthalamic-reticularformationresponsestonoxiousstimulationingoats.Anesthesiology1999,91,1770–1779.[CrossRef]11.Johnson,C.B.;Sylvester,S.P.;Stafford,K.J.;Mitchinson,S.L.;Ward,R.N.;Mellor,D.J.Effectsofageontheelectroencephalographicresponsetocastrationinlambsanaesthetizedwithhalothaneinoxygenfrombirthto6weeksold.Vet.Anaesth.Analg.2009,36,273–279.[CrossRef]12.Murrell,J.C.;Mitchinson,S.L.;Waters,D.;Johnson,C.B.Comparativeeffectofthermal,mechanical,andelectricalnoxiousstimuliontheelectroencephalogramoftherat.Br.J.Anaesth.2007,98,366–371.[CrossRef]13.Orth,M.;Barter,L.;Dominguez,C.;Atherley,R.;Carstens,E.;Antognini,J.Halothaneandpropofoldifferentiallyaffectelectroencephalographicresponsestonoxiousstimulation.Br.J.Anaesth.2005,95,477–484.[CrossRef][PubMed]14.Murrell,J.C.;Johnson,C.B.;White,K.L.;Taylor,P.M.;Haberham,Z.L.;Waterman-Pearson,A.E.ChangesintheEEGduringcastrationinhorsesandponiesanaesthetizedwithhalothane.Vet.Anaesth.Analg.2003,30,138–146.[CrossRef][PubMed]15.Haga,H.A.;Dolvik,N.I.Electroencephalographicandcardiovascularvariablesasnociceptiveindicatorsinisoﬂurane-anaesthetizedhorses.Vet.Anaesth.Analg.2005,32,128–135.[CrossRef][PubMed]16.Diesch,T.J.;Mellor,D.J.;Johnson,C.B.;Lentle,R.G.Electroencephalographicresponsestotailclampinginanaesthetizedratpups.Lab.Anim.2009,43,224–231.[CrossRef]17.Haga,H.A.;Ranheim,B.Castrationofpiglets:Theanalgesiceffectsofintratesticularandintrafunicularlidocaineinjection.Vet.Anaesth.Analg.2005,32,1–9.[CrossRef]18.Waldmann,V.;Otto,K.H.;Bollwahn,W.Pigletcastration-painsensationandpainelimination.Dtsch.Tierarztl.Wochenschr.1994,101,105–109.19.Kells,N.J.;Beausoleil,N.J.;Chambers,J.P.;Sutherland,M.A.;Morrison,R.S.;Johnson,C.B.Electroencephalographicresponsesofanaesthetizedpigs(Susscrofa)totaildockingusingclippersorcauteryironperformedat2or20daysofage.Vet.Anaesth.Analg.2017,44,1156–1165.[CrossRef]20.Kells,N.J.;Beausoleil,N.J.;Sutherland,M.A.;Morrison,R.M.;Johnson,C.B.Electroencephalographicassessmentoforalmeloxicam,topicalanaestheticcreamandcauteryironformitigatingacutepaininpigs(Susscrofa)undergoingtaildocking.Vet.Anaesth.Analg.2017,44,1166–1174.[CrossRef]21.Johnson,C.;Sutherland,M.;Beausoleil,N.;Kells,N.ValidationofEEGMeasuresforPainAssessmentinPigletsAged0to10Days.pork.org.NPB#C-13–188;MasseyUnivesity:PalmerstonNorth,NewZealand,2015.22.Haga,H.A.;Tevik,A.;Moerch,H.Electroencephalographicandcardiovascularindicatorsofnociceptionduringisoﬂuraneanaesthesiainpigs.Vet.Anaesth.Analg.2001,28,126–131.[CrossRef]23.Munglani,R.;Hunt,S.P.Molecularbiologyofpain.Br.J.Anaesth.1995,75,186–192.[CrossRef]24.Hunt,S.P.;Pini,A.;Evan,G.Inductionofc-fos-likeproteininspinalcordneuronsfollowingsensorystimulation.Nature1987,328,632–634.[CrossRef][PubMed]25.Harris,J.A.Usingc-fosasaneuralmarkerofpain.BrainRes.Bull.1998,45,1–8.[CrossRef]26.Coggeshall,R.E.Fos,nociceptionandthedorsalhorn.Prog.Neurobiol.2005,77,299–352.[CrossRef][PubMed]27.Lykkegaard,K.;Lauritzen,B.;Tessem,L.;Weikop,P.;Svendsen,O.LocalanaestheticsattenuatesspinalnociceptionandHPA-axisactivationduringexperimentallaparotomyinpigs.Res.Vet.Sci.2005,79,245–251.[CrossRef][PubMed]28.Svendsen,O.Castrationofpigletsundercarbondioxide(CO2)anaesthesia.J.Vet.Pharmacol.Ther.2006,29,54–55.[CrossRef]29.Nyborg,P.Y.;Sørig,A.;Lykkegaard,K.;Svendsen,O.Nociceptionaftercastrationofjuvenilepigsdeterminedbyquantitativeestimationofc-Fosexpressingneuronsinthespinalcorddorsalhorn.DanskVeterinærtidsskrift2000,83,16–17.30.Ruggiero,D.A.;Sica,A.L.;Anwar,M.;Frasier,I.;Gootman,N.;Gootman,P.M.Inductionofc-fosgeneexpressionbyspinalcordtransectioninSusscrofa.BrainRes.1997,759,301–305.[CrossRef]31.Werner,J.;Saller,A.;Reiser,J.;Senf,S.;Deffner,P.;Abendschön,N.;Fischer,J.;Grott,A.;Miller,R.;Zablotski,Y.;etal.EvaluationofTwoInjectionTechniquesinCombinationwiththeLocalAnestheticsLidocaineandMepivacaineforPigletsUndergoingSurgicalCastration.Animals2022,12,1028.[CrossRef]32.Tierschutzgesetz.TierschutzgesetzinderFassungderBekanntmachungvom18.Mai2006(BGBl.IS.1206,1313),daszuletztdurchArtikel105desGesetzesvom10.August2021(BGBl.IS.3436)geändertwordenist.2021.Availableonline:https://www.gesetze-im-internet.de/tierschg/BJNR012770972.html(accessedon31August2022).33.Sassenhagen,J.;Draschkow,D.Cluster-basedpermutationtestsofMEG/EEGdatadonotestablishsigniﬁcanceofeffectlatencyorlocation.Psychophysiology2019,56,e13335.[CrossRef]34.Akeju,O.;Westover,M.B.;Pavone,K.J.;Sampson,A.L.;Hartnack,K.E.;Brown,E.N.;Purdon,P.L.Effectsofsevoﬂuraneandpropofolonfrontalelectroencephalogrampowerandcoherence.Anesthesiology2014,121,990–998.[CrossRef]

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