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Received:14March2024 Revised:19May2024 Accepted:29May2024 DOI:10.1002/vetr.4395 ORIGINALRESEARCHAntinociceptiveandwoundhealingeffectsofacommercialformulationoflidocaine,bupivacaine,adrenalineandcetrimideappliedtopicallytosuperﬁcialskinwoundsinhorsesShaunPratt1 AlbertSole-Guitart1 KarladeKlerk1 ElizabethEvans2,3 JaneHume2,4 ChiaraPalmieri1 JoanneRainger1 WendyGoodwin11SchoolofVeterinaryScience,UniversityofQueensland,Gatton,Queensland,Australia2Invetus,Casino,NewSouthWales,Australia3Bioproperties,Glenorie,NewSouthWales,Australia4Vetoquinol,Hamilton,Queensland,AustraliaCorrespondenceShaunPratt,SchoolofVeterinaryScience,UniversityofQueensland,Gatton,Queensland,Australia.Email:shaun.pratt@uq.edu.auFundinginformationInvetus,Grant/AwardNumber:2018002127 AbstractBackground:Post-traumaticdistallimbwoundscausediscomfortandhealgraduallybysecondintention.ThetopicalapplicationofTri-Solfen(lido-cainehydrochloride,bupivacainehydrochloride,adrenalineacidtartrateandcetrimide[LBAC])produceseffectivepostsurgicalcutaneousanalgesiainlambs,calvesandpiglets;however,itseffectonwoundsinhorsesisunknown.Methods:Theantinociceptiveeffect,measuredbymechanicalthreshold(MT),andthewoundhealingimpactsofLBACcomparedwithsalinewereinvestigatedonsurgicallycreated20×20mmdistallimbwoundsin10horses.Treatmentwasappliedoncedailyfor7daysfollowingwoundingonday0.Mechanicalthresholdsweremeasuredaftertreatmentondays1,2and3.Healingwasobservedfor25days.Results:ThetopicalapplicationofLBACimmediatelyfollowingwoundinganditsreapplication24hourslaterincreasedtheaverageMTontheﬁrstpost-traumaticdayby3Newtons.However,noantinociceptivebeneﬁtwasobservedondays2or3.TreatmentwithLBACdidnotadverselyaffectwoundhealingwhencomparedwithsaline.Limitations:MethodologicaldifferencesprecludeabsoluteMTcomparisonsbetweenstudies.Theexperimentaldesigndidnotincludeamodelofcontaminatedornaturallyoccurringwounds.Conclusion:LBACmayprovideanearlyantinociceptivebeneﬁtwhenappliedtouncontaminatedsurgicallycreatedwoundswithoutcompromisingheal-ing.KEYWORDSantinociception,mechanicalthresholdtesting,Tri-Solfen,woundhealing,horseINTRODUCTIONTraumaticinjurytothedistalextremitiesofhorsesiscommon.Post-traumaticdistallimbwoundscausediscomfort,arenotoriouslydifﬁculttoman-ageandoftenexhibitprotractedsecond-intentionhealing.Thereisconsiderableinterestindevelop-ingnovelapproachestoimprovethehealingrateoflowerlimbwoundsinhorses,1with,histori- ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution-NonCommercial-NoDerivsLicense,whichpermitsuseanddistributioninanymedium,providedtheoriginalworkisproperlycited,theuseisnon-commercialandnomodiﬁcationsoradaptationsaremade.©2024TheAuthors.VeterinaryRecordpublishedbyJohnWiley&SonsLtdonbehalfofBritishVeterinaryAssociation.cally,astrongfocusonoptimisingthewoundbedmicroclimate.2Theeffectoftreatmenthasbeeninves-tigatedinexperimentalmodelsofsurgicallycreatedwounds2,3andinclinicaltrialsofnaturallyoccur-ringwounds4;however,suchstudieshavefocusedheavilyonwoundhealingwithoutconsideringanalgesia.Providingrelieffromdiscomfortisthecorner-stoneoftraumamanagement.Cutaneouswounds VetRec.2024;e4395.wileyonlinelibrary.com/journal/vetr 1of13https://doi.org/10.1002/vetr.4395 2of13 VETERINARYRECORD causediscomfort,reduceperformance,compro-misethehuman‒animalbondandmayresultinlocalisedhypersensitisationwithassociatedbehaviouralavoidance.5Althoughnon-steroidalanti-inﬂammatoriesareeffectivesystemicanalgesicsforthetreatmentofwound-associatedpaininhorses,clinicallyvalidatedtopicaltherapiesremainscarce.Thisisimportant,as60%ofhorseownersapplypotentiallycytotoxiccompoundstowoundspriortoveterinaryconsultation.6Thisrisksdisruptiontoearlyhaemostaticandﬁbroproliferativecellsandmayreducetheeffectivenessofprospectivetherapiesprescribedbyveterinaryclinicians.Itis,therefore,importanttoprovideownerswithalicencedproductthat(1)deliverseffectiveanalgesiaand(2)preserveswoundhealingpotential.Tri-Solfen(AnimalEthics)isanaqueousﬁxedcombinationproductcontaining40.6g/Llidocainehydrochloride,4.2g/Lbupivacainehydrochloride,24.8mg/Ladrenalineacidtartrateand5.0g/Lcetrim-ide(LBAC).Theproductlabelclaimstoproviderapidanalgesia,visiblyreducebloodlossandimprovewoundhealingwhenappliedtopicallytocutaneouswounds.TheLBACformulationhas36patentsgrantedglobally,includingintheUnitedStates,Europe,NewZealandandGreatBritain(SmithV,MedicalEthics,personalcommunication,2023).InAustralia,LBACisregisteredfortheprovisionofpostoperativeanalge-siafollowinginvasivesurgicalproceduresinlambs,7–9calves10andpiglets.11,12Priortothisstudy,theeffectsofLBAConequinewoundswereunknown.Thisstudywasdesignedasapharmaceuticaltrialtocomparetheantinociceptiveeffects,measuredbymechanicalthreshold(MT)testing,oftopicallyappliedLBACcomparedwith0.9%sodiumchlorideonsurgicallycreateddistallimbwoundsinhorses.TheinﬂuenceofLBACtreatmentonwoundhealingwasasecondaryaimtoensuretheabsenceofadverseeffects.ItwashypothesisedthatLBACwouldprovideshort-durationantinociception(increasedMTs)with-outadverselyaffectingwoundhealing.TheresultsofthisstudyprovidedtheevidencethatledtotheLBACformulation,marketedasTri-Solfen,receiv-ingregistrationwiththeAustralianTherapeuticGoodAssociationasanon-prescriptionveterinaryproductforuseonpost-traumaticequinewounds.MATERIALSANDMETHODSAnimalsAllprocedureswereperformedwiththeapprovaloftheUniversityofQueensland’sProductionandCompanionAnimalEthicsCommittee(SVS/300/18/INVETUS).Tenhorses(ninegeldingsandonemare)aged7.3±3years,belongingtotheUniversityofQueensland,wereenrolledinthestudy.ThepopulationincludedfourStandardbreds,oneStandardbredcrossandﬁveThoroughbreds.Allani-malswereconsideredhealthybasedonaphysicalexaminationandmetthefollowinginclusioncrite-ria:morethan2yearsofage,indateHendravirusandtetanusvaccination,easyandsafetohandle,nolamenessoridentiﬁableskinlesionspresent,7dayswithheldfromallmedications(includingnon-steroidalanti-inﬂammatories)andnoknownpreviousadversereactionstolocalanaestheticsorinjectablesedatives.Atthecompletionofthestudy,allani-malswerereturnedtotheUniversityofQueensland’steachingandresearchherd.SummaryofstudydesignThestudywasaprospective,randomised,blinded,longitudinalcontrolledstudy.ThestudywasdesignedandexecutedaspertheCONSORTguidelines.13Fol-lowingthecreationofbilateralsurgicalwoundsonthethirdmetacarpi,eachlimb(leftorright)wasran-domisedusingacointosstoreceivetopicaltreatmentwitheitherLBACor0.9%sodiumchloride(saline).Theantinociceptiveandwoundhealingeffectswereassessedbyveterinarianswhowereblindedtothetreatmentallocation.Afulldescriptionofthemechan-icalstimulusisprovidedbelow;however,earlierworkinhorses,usinganidenticalcontrolunitandpneu-maticactuatorconﬁguration,deﬁnedtheMTofintactepidermisas3.1±2Newtons(N).14Todetect3NofdifferenceinMTsbetweentreatments,atotalofeightwoundswerethereforerequiredpertreatmentgroup(α=0.05andβ=0.8).TreatmentTheLBACtreatmentcontained40.6g/Llidocainehydrochloride,4.2g/Lbupivacainehydrochloride,24.8mg/Ladrenalineacidtartrateand5.0g/Lcetrim-ideinaﬁxedcombinationsolutionthatwasblueincolour.Tomaintainblindingtotherelevantstudypersonnel,thesalinecontrolsolutionwassimilarlycolouredbyadding2.5mLofbluedye(QueenBlueFoodColour)containing1.8%totaldyestuffto250mLof0.9%sodiumchloride(BaxterHealthcare).Identi-calspraybottleswereusedtoapply1mLtopically(2×0.5mLspray)ofeachsolutiondirectlyontotheallocatedwoundarea(1mLper20mm2).Thiswassufﬁcienttocovertheentireareawithminimalrunoff.Bothsolutionswerestoredinaninsulatedboxinatemperature-controlledroom(<25 C)whennotinuse.MechanicalthresholdtestingAwirelessMTtestingsystem(WMT2LargeAni-mal,TopcatMetrology)wasusedforthestudy.TheapplicationofthedevicewasidenticaltopreviousdescriptionsbyTaylor.15Brieﬂy,themechanicalstim-uluswassuppliedbypneumaticactuators,whichdroveanearlyhemispherical1mmpinattachedto VETERINARYRECORD 3of13 FIGURE1(a)Thecontrolunitandreservoirdevicemountedoverthehorse’swithersandsecuredwithVelcrotoalightweightbellystrap.Pressuretubingconnectsthecontrolandreservoirdevicetothepneumaticactuatorﬁttedtoeachmetacarpus.(b)Thepneumaticactuatorsﬁttedtoeachmetacarpusandsecuredwithmodiﬁedbrushingbootsamovingpistonmountedonarollingdiaphragm.Thepneumaticactuatorswereﬁttedtotheanterioraspectofeachmetacarpusandwereheldinplacebymodiﬁedbrushingboots.Thecontrolunitwasmountedoverthehorse’swithersandsecuredwithVelcrotoalightweightbellystrap.Arechargeablepres-surereservoir(60mLsyringe)wasmountedalongsidethecontrolunitandwasconnectedviapressuretub-ingtothepneumaticactuatorunits(Figure1).AsinglemodiﬁcationtotheMTdeviceinvolvedtheadditionofasmallsiliconcoveringatopthe1mmpin.Thisapproachaimedtopreventlocalisedtissuetraumaassociatedwithrepeattesting.Thesiliconcoveringﬁt-tedtightlyandfollowedtheroundedcontourofthepinhead.Thecontrolunitsequentiallyincreasedtheforcegeneratedbytheactuatorsthroughthe1mmpinatarateincreaseof0.8N/second,from0to20N.Thetest-inglimitwassetat20N.TheoverallMTforeachlegwasrecordedasthemeanofatleasttwotestsrecordedwithin2Nofeachother.Thetestingintervalwas5minutes.Bothactuatorswereﬁtted;however,onlyonelegwastestedatatime.Apositiveresponse(indicatingMT)wasdeﬁnedastheliftingofalegorfoot,pawingattheground,stampingafoot,ﬂexingtheantebrachiumorwalkingtoavoidstimulus.16AtallMTcollectionpoints,theoperatorremainedoutsidethestallandthehorsewasfreetomoveaboutitsstall.Testingbeganwheneachhorsewasvisiblyrelaxedandnotdistractedbyenvironmentalstim-uli.Insectrepellent(N-diethyltoluamide,di-N-propylisocinchomeronate,N-octylbiccloheptenedicarobox-imide,piperonylbutoxide,citronella,pyrethrinsolu-tion;Repel-XTroyLaboratories)wasusedtominimiseconfoundingbehaviourduringMTtesting.TolimitthelearningeffectassociatedwithrepeatMTtest-ing,alleffortsweremadetominimisehorseroutineandnotestingcueswereused.Additionally,irrespec-tiveoftreatmentallocation,odd-numberedhorsesunderwentMTtestingoftheleftlegﬁrst,whileeven-numberedhorsesunderwenttestingoftherightlegﬁrst.ExperimentalprotocolAﬂowdiagramdepictingtheexperimentalproto-colisshowninFigure2.Inthe7dayspriortotheexperimentalphase,eachhorsewasweighed,assignedanumberedtagandunderwentafullclini-calandlamenessexamination.Animalsenteringtheexperimentalphase(startingfromday−1)wereran-domised,groupedandstaggered7daysapart.Horsesweresequentiallynumberedastheyenteredthestudy(horses2‒11).Horse1wasapilotanimalthatdidnotcontributetothestatisticalanalysis.Day–1:acclimatisationPairedhorseswerebroughtinfromtheirpaddocksandhousedinshelteredstallsmeasuring5×5m.Foracclimatisationpurposes,eachhorsewasﬁttedwiththeMTdeviceinanidenticalfashiontohowitwouldbeﬁttedfortesting.Thedevicewasremovedafter20minutes.NoMTtestingwasperformed.Day0:baselineMTtesting,surgicalwoundingandtreatmentapplicationBaselineMTtestingonintactepidermiswasper-formedpriortosurgicalwoundingwiththedeviceﬁttedaspreviouslydescribed.Toreducecross-contaminationbetweenhorses,thetestingsitewascoveredwithanantimicrobialincisedrape(3MIoban2AntimicrobialInciseDrape6640EZ,3M).FollowingbaselineMTdatacollection,horseswererestrainedinstockswithcrosstiesandsedatedwith0.01mg/kgintravenous(IV)detomidine 4of13 VETERINARYRECORD FIGURE2Timelineoftheexperimentalprotocol.Animalsenteringtheexperimentalphase(startingfromday−1)wererandomised,groupedandstaggeredsevendaysapart.Mechanicalthreshold(MT)datawerecollectedinstalls,whilewounding,treatmentapplicationandwoundassessmentwereperformedinstocks FIGURE3Anexampleofsurgicallycreatedbilateralthoraciclimbwoundsonthedorsal45 medialaspectofthemid-thirdmetacarpi.Theimageisofhorse8onday0hydrochloride(EqusedanVet,Ausrichter).Fivemin-uteslater,thesurgicalsiteonboththoraciclimbswasclippedandpreparedusingacombinationof5%chlorhexidinescruband70%alcoholspray.Aringblockusing10mLof2%lidocaine(Lignocaine20,TroyLaboratories)wasperformedproximallytothesurgicalarea.Asterile,autoclavedstencilandrulerwereusedtoreproducesurgicalwoundsinallhorses.Afull-thickness20×20mmsurgi-calwoundwascreatedonthedorsal45 medialaspectofthemid-thirdmetacarpiusinganumber10scalpelblade(Figure3).Thesurgicalsectionswereﬁxedin10%neutralbufferedformalinwithatissue-to-formalinratioof1:10ormore.Toreduceexcessivehaemorrhage,thewoundswereinitiallycov-eredwitha5×5cmlowadherent,sterileabsorbentdressing(Cutilin,Smith&Nephew).After2hours,theabsorbentdressingwasremovedandthetreat-ment(LBACorsaline)wasappliedtotheallocatedwound.Once30minuteshadelapsed,thetreatedwoundswerebandagedwithanabsorbentdressingandalayerofadhesivebandage(AskinaPlastE,BBraun)andcoveredwithcommerciallyavailablesta-blewraps.Horseswerereturnedtotheirstalls.Nofurtherexperimentalprocedureswereperformedonday0.Days1‒3:antinociceptiveMTtestingpriortoandfollowingtreatmentapplicationThestablewrapsanddressingswereremovedeachday,andanantimicrobialincisedrapecuttosuf-ﬁcientlycoverthewoundwasappliedtoprotectthetissuefromtheMTdeviceandtominimisecross-contaminationbetweenwounds.PretreatmentMTtestingwasperformedwithhorsesinstalls,asdescribedpreviously.Theantimicrobialincisedrapewasthenremoved,andhorseswererestrainedinstocksfortreatmentreapplication.EachhorsewastreatedwithLBACorsalineontheallocatedwoundatthesametimeeachday(±15minutes)bythesametwounblindedstudypersonnel.Thetreatedwoundswereleftuncoveredfor20minutesbeforeanewantimi-crobialincisedrapewasapplied.Post-treatmentMTtestingwasperformedwithhorsesfreeinstallsat0.5,1and3.5hoursfollowingtreatmentapplication.Theactuatorpinofthepneumaticdevicewithitssiliconcoveringwascentredwithinthedorsolateralquadrantofthewoundedarea.Oncetestingwascom-plete,theantimicrobialincisedrapewasremovedandthewoundswererebandagedaspreviouslydescribed. VETERINARYRECORD 5of13 Days4‒6:treatmentapplicationonlyTreatmentcontinueddaily.NoMTtestingwasper-formed.Days7‒25:turnoutOnday7,alldressingswereremoved,andthehorseswereindividuallyturnedoutintoa12×6mgrassypaddockuntilday25.Day25:histopathologysamplingOnday25,thehorseswereagainrestrainedinstocksandsedatedwith0.01mg/kgIVdetomidinehydrochloride.Once5minuteshadelapsed,theskinwassurgicallypreparedandaringblockwasper-formedusinglidocaineaspreviouslydescribed.An8mmbiopsypunchwastakenfromthedorsome-dialwoundmarginandﬁxedin10%neutral-bufferedformalin.Histopathologicalanalysisofthepairedsamples(days0and25)wasperformedbyablindedveteri-narypathologist.Tissuesampleswereembeddedinparafﬁn,sectionedatathicknessof5µmandstainedwithhaematoxylinandeosinandcollagen-speciﬁcMasson’strichromestaining.ThehistologicalcriteriaforwoundhealingwereextrapolatedfromTheoretetal.17Brieﬂy,sectionsoftissuewereevaluatedforthepresenceorabsenceofsurfaceulceration,gran-ulationtissue,keloidalcollagen,haphazardcollagenﬁbrearchitecture,ﬁbroblastinﬁltrationandﬁbrinoidnecrosisofthedermalvessels.Thedegreesofder-malvascularity,inﬂammation,ﬁbrosis,haemorrhage,inﬁltrationofeosinophilsandﬁbroblastcellularityweregradedcategoricallyfrommildtosevere.Finally,acomparativestageofhealingatday25wasassessedbetweentreatments.WoundassessmentWoundswerephotographedondays0,4,11,18and25.Thewoundareawasmeasuredusingimageanaly-sissoftware(ImageJ,USNationalInstitutesofHealth)witha60×60mmrulerheldadjacentforstandardref-erence(Figure4).Theareaofeachwoundinmm2wasmeasuredthreetimesandtheaveragewascalculatedtomakeasinglemeasurementforeachday.Thiswasusedtocalculatethewoundarea(mm2)andtherateofhealingdependentontheelapsedday.Rateofhealingdependantonelapsedday=−1 woundarea2−woundarea1 time2−time1 mm2 day Additionally,thephotographswereusedtogradetheamountofmacroscopicgranulationtissuepresentondays4,11,18and25accordingtothefollowing FIGURE4Anexampleofwoundassessment(horse8onday18).A60×60mmtemplatewasusedforstandardreferenceduringphotographicassessmentofwoundhealing.Imagesweremeasuredusingimageanalysissoftware(ImageJ)standards:gradeI,depressedbelowtheskinedge;gradeII,proliferatedtotheleveloftheskinedge;gradeIII,elevatedabovetheskinedge;andgradeIV,requiredtrimmingbacktotheleveloftheepitheliumtocontrolexcessiveproliferationthatinhibitedtheadvancementoftheepithelium.GradeIVgranulationtissuewasconsideredexuberant.18Allmacroscopicgradingswereperformedbythesameblindedindi-vidualinrandomsuccessionatthecompletionofthestudy.GeneralhorsewelfareFreshwaterwasprovidedadlibitumandlucerneorgrassyhaywasprovidedtwicedailythroughoutthestudy.Afterday7,horsesalsohadaccesstograss.Dailyexaminationsrecordedthedemeanour,eating,drinking,urinating,defecationandgeneralbehaviourofallanimalsfromday−1today25.Ondays0‒3,allhorsesunderwentaphysicalexaminationandwerepainscoredbyaveterinarianblindedtotreatmentapplicationusingavisualanaloguescale(VAS).TheVASwasa100mmlinewith0representingnopainand100representingimmensepain.IfanyhorsescoredaVASof40mmormore,rescueanalgesia(methadone0.1mg/kg)andsedation(acepromazine0.01mg/kg)wereadministeredIV.Horseswerereassessedafter30minutesandadministeredﬂunixinmeglumine1.1mg/kgIViftheVASscoreremained40mmorgreater.Horsesthatreceivedrescueanalgesiawereexcludedfromthestudy.StatisticalanalysisThedatawerecomparedusingeitherGenStat(Release22.1,VSNInternational)orTIBCOSpotﬁreS+8.2 6of13 VETERINARYRECORD (2010).Descriptivesummariesarepresentedasthemean±standarderrorofthemean.Mechanicalthresholdmeanswerecomparedusingalinearmodelwiththefollowingﬁxedvariables:treatment(LBAC,saline),day(1,2,3),timepointwithinday(0.5,1,3.5hours),replicateandtestleg(leftorright).Themodel’ssuitabilitywasassessedusingplotsofresiduals.Addi-tionally,MTdatawerecomparedwithineachdayusingthesamemodel(withdayremoved)andsuit-abilityassessment.Signiﬁcancewassetatap-valueoflessthan0.01.Woundhealingdata,includingwoundareaovertimeandwoundhealingrate,werecomparedusingalinearmodelwiththefollowingﬁxedvariables:treatment(LBAC,saline),day(0,4,11,18,25)andtestleg(leftorright).Themodel’ssuitabilitywasassessedusingplotsofresiduals.Additionally,woundareaandwoundhealingratewerecomparedwithineachdayusingthesamemodel(withdayremoved)andsuit-abilityassessment.Signiﬁcancewassetatap-valueoflessthan0.05.RESULTSNoVASscorewasabove0foranyhorse,andnores-cueanalgesiaorsedationwasprovidedtoanyhorse.Onehorsedevelopedwhitelinediseaseintherightthoracicfootonday13(LBAC-treatedlimb).Thehorsewassoundatthetrotandnegativetohooftesters.Thehorsewastreatedwithcorrectiveshoeingandapoultice.ThisanimalwasnotexcludedfromthestudybecauseMTtestinghadalreadyconcluded.Onehorse(horse3)waswithdrawnfromthestudyonday1duetoabnormalitiesonphysicalexamination(tachyp-noeaandventraloedema).Thehorsewasdiagnosedwithachronicdiseaseunrelatedtothestudyanddidnotcontributetostatisticalanalysis(n=9).MechanicalthresholdtestingTwotothreereplicateMTmeasurementsweresuf-ﬁcienttogeneratereproduciblereadings(<2N)forallanimals.TheaveragemechanicalforcerequiredtoelicitabehaviouralresponsepriortowoundingwassimilarbetweentheLBAC(4.2±0.6N)andsaline(4.2±0.5N)groups.TheaverageMTmeasuredatbaselineandfollowingtreatmentreapplicationondays1,2and3isillustratedinFigure5.ThelinearpredictivemodelfortheMTdataisshowninTable1.Overall,treatmentwithLBACincreasedtheaverageMTrequiredtogenerateabehaviouralresponsewhencomparedwithsaline(p<0.001).Themostsigniﬁcanttreatmenteffectwasseenonday1(p=0.003).Onday1,theaver-ageincreaseinMTfollowingtreatmentwithLBACwas3.0N(minimum1.0Nandmaximum5.0N).Thiseffectwasnotinﬂuencedbytime(timepointp=0.441),indicatingthat,comparedwithsalinetreatment,LBACtreatmentsigniﬁcantlyincreasedtheaverageMTatalltimepoints(0.5,1and3.5hours)onday1(Figure5).Onday2,treatmentwithLBACdidnotaltertheMTcomparedwithsaline(p=0.131).Onday3,theaverageincreaseinMTfollowingtreatmentwithLBACcomparedwithsalinewas2.3N(minimum0.5Nandmaximum4.0N)(p=0.011);however,thiseffectwassigniﬁcantlyinﬂuencedbytime(timepointp=0.004)andfellbelowthepowerofdetectionandstatisticalsigniﬁcanceforthestudy.WoundhealingLimbwoundsrandomisedtoreceivetreatmentwithLBACwereinadvertentlycreatedlargerthanthoseran-domisedtoreceivetreatmentwithsaline(LBAC:360±12mm2andsaline:342±10mm2).Theareaofallwoundsincreasedonday4beforereducingondays11,18and25(Figure6).Comparablehealingrateswerefoundbetweentreatmentsoverthestudyperiod(Figure7).Thelinearpredictivemodelfoundnodifferencebetweentreatmentswithrespecttohealingrate(p=0.717)orwoundarea(day0,p=0.281;day4,p=0.555;day11,p=0.244;day18,p=0.119;day25,p=0.131).HistopathologyandgrosstissueassessmentForbothtreatmentgroups,thesurgicalsectionscol-lectedonday0wererepresentativeofhealthytissue.Histopathologicalanalysisofbiopsysamplescol-lectedonday25revealedthatfourwoundstreatedwithLBACwereslightlyimmaturewhencomparedwiththoseofthecontralaterallimbtreatedwithsaline,twowoundstreatedwithsalinewereslightlyimmaturewhencomparedwiththecontralaterallimbtreatedwithLBACandthreewoundswereincomparablestagesofhealing(Table2).Overall,allwoundsinbothtreatmentgroupsexhibitedheal-ingprogressionthatwasappropriateforthestudydurationandwoundtype.Thecompletehistopatho-logicalgradeforeachwoundisavailableinthesupportinginformation.Noexuberantgranulationtissue(EGT)formedwithinanywoundtreatedwithLBACorsaline(Table3).Allwoundsexhibitedgranulationtissuebelowtheskinedgesonday4.Ondays11,18and25,sixLBAC-treatedandsixsaline-treatedwoundswerescoredasgradeIII(Figure4)and16LBAC-treatedand14saline-treatedwoundswerescoredasgradeII.DISCUSSIONThisstudyusedMTtestingtoinvestigatetheantinoci-ceptivepropertiesofacommercialformulationofLBACappliedtopicallytosurgicallycreatedfull-thicknesswoundsonthedistalthoraciclimbsofhorses.TreatmentwithLBACfollowingwoundinganditsreapplication24hourslaterincreasedtheaver-agemechanicalforcerequiredtoelicitabehaviouralresponse.Thepeakantinociceptiveeffectappearedto VETERINARYRECORD 7of13 FIGURE5Averagemechanicalthresholds(MT)measuredfromninehorsestreatedwitheitherlidocaine‒bupivacaine‒adrenaline‒cetrimide(LBAC)orsalineappliedtopicallytosurgicallycreateddistalthoraciclimbwounds.MTwasmeasuredonday0priortowounding(baseline)andoneachconsecutivedaypriortotreatment(0)andat0.5,1and3.5hoursfollowingtreatment.Thedataareexpressedasthemean±standarderrorofthemean.Asteriskindicatesstatisticalsigniﬁcanceextrapolatedfromthelinearpredictivemodel(p<0.01)TABLE1Treatmentwithlidocaine-bupivacaine-adrenaline-cetrimide(LBAC)orsalineappliedtopicallytosurgicallycreateddistalthoraciclimbwoundsinninehorses.Averagemechanicalthresholdsbetweentreatmentswerecomparedusingaﬁxedeffectslinearmodel. ParameterFstatisticp-ValueLower95%CI(N)Pointestimateofdifference(N)Upper95%CI(N) OverallTreatment17.78<0.0011.22.23.3Day4.490.012Timepoint2.270.079Replicate0.020.880Testleg0.160.685Day1Treatment8.930.0031.03.05.0Timepoint0.900.441Replicate0.010.918Testleg0.240.626Day2Treatment2.310.13101.43.2Timepoint0.440.725Replicate0.010.929Testleg1.070.303Day3Treatment6.690.0110.52.34.0Timepoint4.650.004Replicate0.090.768Testleg0.050.828 Abbreviation:CI,conﬁdenceinterval.Boldingwasusedtohighlightthestatisticallysigniﬁcantvaluesinthetable.occur0.5‒1hourafterthereapplicationofLBAConday1(24hoursafterwoundcreation).Noapprecia-bleantinociceptivebeneﬁtwasdetectedfollowingthereapplicationofLBAConday2or3(48and72hoursafterwoundcreation).Additionally,theuseofLBACappliedtopicallyoncedailyfor7dayshadnoadverseeffectsonwoundhealingoverthe25-daystudyperiod.NociceptivethresholdtestingNociceptivethresholdtesting(NTT)isarecognisedanalgesiometrictechniqueusedtoinvestigatetheefﬁcacyofanintervention.5Itinvolvesapplyinganox-iousstimulus(mechanical,electricalorthermal)inanintensifyingmanneruntilthedesiredbehavioural 8of13 VETERINARYRECORD FIGURE6Changesinwoundareainninehorsestreatedwitheitherlidocaine‒bupivacaine‒adrenaline‒cetrimide(LBAC)orsalineappliedtopicallytosurgicallycreateddistalthoraciclimbwounds.Measurementsweremadeusingimageanalysissoftware(ImageJ)andareexpressedasthemean±standarderrorofthemean FIGURE7Woundhealingrateforninehorsestreatedwitheitherlidocaine‒bupivacaine‒adrenaline‒cetrimide(LBAC)orsalineappliedtopicallytosurgicallycreateddistalthoraciclimbwounds.Measurementsweremadeusingimageanalysissoftware(ImageJ)andareexpressedasthemean±standarderrorofthemeanresponseiselicited.16Thedifferenceinnociceptorthresholdspriortoandfollowingtheapplicationoftheinterventioninfersitsantinociceptiveeffect.MTtestingisaneffectivemethodtoquantifythenocicep-tivethresholdinnon-verbalanimals.19Whenusedasanon-invasivenociceptivechallengetostudywound-associatedpaininhorses,MTtestingwasmoresen-sitiveandspeciﬁcthanelectricalorthermaltesting.14Comparedwiththermalthresholdtesting,MTtest-inggeneratesareproducibleforcestimulus,canberemotelycontrolledandcarrieslesslocalisedtissuetrauma.14However,therearelimitationstoMTtest-ing.Theseincludeconditioningbiasandrepeattestingtrauma.20,21Withrepeattesting,horsesmayassociatetheliftingofalegwiththereleaseofpressureandmayproceedtolifttheirlegatprogressivelylowerthresholds.Thisresultsinatemporalshiftinthethresholddataandistermedlearningbias.Similarly,horsesmayassociatethetestingapparatusortestingcueswithimpendingnoxiousstimuli.20,21TheadvantagesofthewirelessMTdevicewereitslightweightattachmentsanditsportablehandheldtransmitter.Thisallowedallper-sonneltoremainoutsidethestallduringtestingandminimisedtheamountofequipmentrequired.Testingcueswereomitted,andtheorderoflimbstestedwasrandomised.ThereareimportantconsiderationswithinthemethodsthatneedtobeaddressedinrelationtotheMTresults.First,toreducetheincidenceofrepeattestingtrauma,asiliconecoveringwasplacedoverthe1mmpin,whichwasdrivenbythepneumaticactuators,duringallMTdatacollectionpoints,includ-ingbaseline.Topreventwoundsitecontaminationandcross-contaminationbetweenhorses,allwoundswerecoveredwithathinantimicrobialincisedrapeduringtesting.TheseadjustmentslikelyaccountedfortheonaveragegreaterMTrecordedpriortowounding(intactepidermis)comparedwithaprevi-ousreport.14Intranslationalhumanstudies,probesofsmallercross-sectionalareareducescatterwithinMTtesting,22suggestingthattheadditionofasili-concapmayalsoexplainthevariabilityinthepresentdata.TheonaveragegreaterMTonintactepider-mismaycausethepowercalculationtounderesti-matetherequirednumberofwounds.Toaccountforthescatterandpotentiallyreducedstatisticalpower,thep-valueforMTsigniﬁcancewassettolessthan0.01.Finally,itisimportanttoconsiderthedifferencebetweenpressureandforce.Pressureequatestotheforceappliedperunitarea.Anincreaseinsurfaceareabytheadditionofthesiliconcapmayreducepres-suretransfertoindividualperilesionalnociceptors,yetmayincreasethetotalnumberofperilesionalnocicep-torsstimulated.Thenon-linearrelationshipbetweenprobediameterandresultsfromMTtestingisshownbyTayloretal.23Thismodiﬁcationpreventsthedirectcomparisonofresultswithotherstudies;however,theauthorsbelievethattheconservedmethodology VETERINARYRECORD 9of13 TABLE2Summarisedhistopathologicalanalysisofsurgicallycreateddistalthoraciclimbwoundsinninehorsestreatedtopicallywitheitheracommercialformulationoflidocaine‒bupivacaine‒adrenaline‒cetrimide(LBAC)orsaline LBACSaline Day0Day25Day0Day25(n) DermalvascularityNoincreaseorminimal9090Mild0000Moderate0706Severe0203DermalinﬂammationAcute08a07Chronic02a02DermalinﬂammationAbsent9190Minimal0000Mild0202Moderate0403Severe0204DermalﬁbrosisAbsent9090Minimal0000Mild0302Moderate0607Severe0000FibroblastcellularityNoincreaseorminimal9090Mild0000Moderate0807Severe0102DermalhaemorrhageAbsent9090Mild0307Moderate/severe0602InﬁltrationofeosinophilsintothedeepdermisAbsent9090Minimal0104Mild0605Moderate/severe0200SurfaceulcerationAbsent9191Present0808GranulationtissueAbsent9090Present0909KeloidalcollagenAbsent9999Present0000HaphazardcollagenﬁbrearchitectureAbsent9897Present0102 (Continues)TABLE2(Continued) LBACSaline Day0Day25Day0Day25(n) FibroblastsAbsent0000Present9999FibrinoidnecrosisofthedermalvesselsAbsent9798Present0201 Note:Valuesrepresentthenumber(s)ofhorsesﬁttingeachcategory,withthehighestpossiblenumberbeinganine.aOnewoundtreatedwithLBACdisplayedindicatorsforacuteandchronichaemorrhageonday25.remainssufﬁcienttoconcludeontherelativeeffectofLBACwithinthemodeldescribed.NoxiousthresholdtestingbyMThasbeenval-idatedonlyonintactepidermis.Previousstud-ieshaveappliedmechanicalstimulustotheaxialmusculature,24,25face,26regionsofthefoot27,28andmetacarpus29–32toinvestigatetheantinociceptivepropertiesofvariouscompoundsinhorses.TheextrapolationofMTdatacollectedfromintactder-misinsuchstudiesiscautionedduetodifferencesinpatientsensitisationandtheremovalofdermalnoci-ceptorsfollowingwounding.Full-thicknesswoundsareexpectedtoremovenociceptorterminalswithinthedermisandexposetheunderlyingfatandextracel-lularmatrixoftheinterstitium.Therefore,stimulationofnociceptorslocatedwithintheperilesionalder-misisresponsibleforthetransmissionofnociceptionincutaneouswoundmodels.Shalloworpartial-thicknesswoundsmaybemorepainfulthanwoundsthatpenetratethedeepermusculatureduetothehighdensityofnociceptorswithinthesuperﬁcialdermis.Forexample,thelikelihoodofdevelopingmoderatetoseverewound-associatedpaininhumansis2.7timesgreaterifthewoundispartiallythick.33Consequently,otherformsofNTTthataremorespeciﬁcintheirsomatosensorylocalisationtothewoundperipherywouldlikelygeneratethresholdsofdifferingmagni-tudes.VariableMTsmayalsobeexpectedifthesamedevicewastestedonpartial-thicknesswounds.TheeffectofLBAContheMTwasnotinvesti-gatedonthedayofwoundingasitwashypothesisedthattheeffectoftreatmentmaybedifﬁculttosep-aratefromresidualanalgesiaprovidedforstandingsedation.FailuretotestonthedayofwoundingmayhavepretermittedthetruepeakantinociceptiveeffectofLBAC.ThemarginalincreaseinMTmeasuredonday3mayrepresentmeasurementdriftinthedevice,atype1statisticalerror,localtissuesensitisation21orsimplyachangeindermatomesensitivitywithinthegranulatingtissuemargin.Partitioningthephasesofwoundhealingintodiscretetemporalcategories(haemostasis,inﬂammatory,proliferativeandremod-ellingphase)isanoversimpliﬁcationofthecomplexanddynamicbiologicalprocessesresponsibleforwoundclosure;however,earlyﬁbroproliferationmay 10of13 VETERINARYRECORD TABLE3Macroscopicgranulationtissuerecordedondays4,11,18and25inninehorsesfollowingtopicaltreatmentofsurgicallycreateddistalthoraciclimbwoundswitheitheracommercialformulationoflidocaine‒bupivacaine‒adrenaline‒cetrimide(LBAC)orsaline LBACSaline Day4Day11Day18Day25Day4Day11Day18Day25 GradeI92119313GradeII06560464GradeIII01320222GradeIV00000000 Note:Valuesrepresentthenumber(s)ofhorsesﬁttingeachcategory,withthehighestpossiblenumberbeinganine.havecontributedtothedeclineinantinociceptiveeffectbyday2.AmajorchallengetoNTTisthevariabilityinbehaviouralresponsestoidenticalnoxiousstimulibetweenanimals.Painisanunpleasantsensoryandemotionalexperienceassociatedwithactual,potentialorperceivedtissuedamage.Paininformationcon-nectsviacomplexneurobiologicalpathwaystoaltertheanimal’saffectivestate(behaviour).Forethicalrea-sons,allhorsesreceivedmultimodalanalgesiaatthetimeofsurgicalwounding,includingsubcutaneouslidocainehydrochlorideprovidedasaringblockandtheIVadministrationofdetomidinehydrochloride.Thepre-emptivemodulationofnociceptionintheseanimalstoachievestandingsedationmaynotappro-priatelymodelacutelytraumaticwoundscenarios.TheresultsfromthecurrentstudyindicatethatLBACmaybeeffectiveatincreasingtheMTofsurgicallycre-atedfull-thicknesscutaneouswounds;however,vari-ableresponsesmayoccurinpatientswithtraumaticwoundsorchronicallyinﬂamedwounds.NoxiousthresholdtestingwastheprimaryoutcomemeasureusedtoinvestigatetheantinociceptiveeffectsofLBAConfull-thicknesscutaneouswounds.TheVASwasusedtoassessoverallhorsewellbeingandtodeterminewhethersystemicanalgesicinterventionwasrequiredtotreatpain.Inarecentinvestigation,theVASdisplayedgoodtoverygoodrepeatability,goodtoverygoodreproducibilityandmoderatetogoodvaliditycomparedwiththemultidimensionalCom-positeOrthopaedicPainScale,whenpainwasgradedbyaveterinarian.34TheoverallsensitivityoftheVAStodetectpaininhorsesfollowingundisclosedproce-dureswas96%.34Unfortunately,avalidatedpainscale(‘goldstandard’)iscurrentlyunavailableinhorses.Toensurethathorsewelfareisprioritised,additionalmultidimensionalpainscalesshouldbeconsideredinfuturestudies.WoundhealingComparedwiththoseoftrunklesions,woundsonthedistallimbsofhorsesundergoaprolongedandoftenineffectualacuteinﬂammatoryphase,char-acterisedhistologicallybylownumbersofpoorlyarrangedmyoﬁbroblastsandfewpolymorphonu-clearcells.35,36Suchwoundsappeartorelymoreoncontracturethanre-epithelisationforsecond-intentionhealing.37ThisprocessisimpededbyEGT.ManyfactorsareimplicatedinEGTformation,forexamplelowoxygentension,poorbloodsupply,animbalanceinpro-inﬂammatorycellularsignallingmolecules(Transforminggrowthfactorbeta1(TGF-B1)andHypoxia-induciblefactor1-alpha(HIF-1a))2andthepersistenceofhaphazardlyarrangedﬁbrindepositions.35Despitegranulationtissuebeingaprominenthistologicalfeatureinallwoundsonday25(LBAC9/9andsaline9/9),thehaphazardarrangementofcollagenﬁbreswasidentiﬁedinonlyoneoftheninewoundstreatedwithLBACandtwooftheninewoundstreatedwithsaline(Table2).Additionally,nogradeIVmacroscopicgranulationtissuewaspresent(Table3),andallwoundswereatanacceptablestageofhealingbyday25(TableA1).ThelowincidenceofEGTislikelytheresultofstudydesign.Allwoundswerecreatedasepticallyandremaineduncontaminatedthroughoutthestudy.EGTisacommoncomplicationduringthemanagementoflowerlimbwoundsinhorsesduetothehighrateofwoundcontamination.2Thepresenceofwoundcontaminationappearstobeanimportantfactorwheninvestigatingnoveltopicaltherapies.Extensiveworkbythesameresearchgroupdocumentedthevariabilityinhealingratesbetweencontaminated38anduncontaminated39woundstreatedwithvariouspreparationsofmanukahoney.Therefore,itisrea-sonabletosuggestthattheeffectsofLBACmaydifferifappliedtocontaminatedwounds,anditremainsunclearwhetherLBACpreventstheformulationofEGT.ThisisacommonshortcominginexperimentalmodelsinvestigatingEGTformationandisdiscussedindetailbyAnantamaetal.1Thevolumesusedforsalinetreatmentwereinsuf-ﬁcienttolavagethewoundorsigniﬁcantlyalterthemicroclimatepH,meaningthattheeffectsofsalineasadecontaminantwerelikelyminimal.However,inadditiontothepre-emptivenociceptivemodu-lationbydetomidinehydrochlorideandlignocainehydrochloride,salinetreatmentmayhavereducedthepotentialdifferenceinantinociceptiveandwoundhealingeffectsbetweenLBACandsaline.Thismayreducethepertinenceofthepowercalculation.Localanaestheticsareextensivelyusedinveteri-naryhospitalstoprovidelocaltissuedesensitisationpriortoandfollowingsurgicalintervention.Despiteclaimsofreducedtensilestrengthandincreasedleukocytictrafﬁckingfollowingtheuseofsomelocalanaesthetics,datafrommurineandlagomorphmodelsindicatethatlidocainehydrochlorideand VETERINARYRECORD 11of13 bupivacainehydrochlorideresultinnoadverseeffects.40–42However,thetopicaluseoflido-caine‒adrenalineislesswelldeﬁned.Inrats,thesubcutaneousinﬁltrationoflidocaine‒adrenalinetransientlydelayedwoundhealingonday7andresultedinthedelayedrearrangementofcollagenﬁbres.43AsimilarphenomenonmayhaveoccurredinwoundstreatedwithLABC,withratesofhealingretardingonday11beforeacceleratingtowardsday25(Figure7).Anincreaseinwoundareabetweendays0and4isknowntooccurduringtheearlystagesofwoundhealingduetothecontractionofperilesionaltissue.Theeffectofcetrimideonﬁbroblastviabilityandwoundhealinghasnotbeeninvestigated.Medi-Solfen,asimilarproducttoTri-Solfen,containing5%(w/w)lidocainehydrochloride,0.5%(w/w)bupiva-cainehydrochloride,0.00451%(w/w)adrenalineacidtartrateand0.5%(w/w)cetrimide,iscurrentlyunder-goingphasetwoclinicaltrialsforuseonacutelytrau-maticskinwoundsinhumans.Theresultsfromtheseclinicaltrialsmayprovideadditionalinformationinthefuture.Onehorsedevelopedwhitelinediseaseonday13duringtheturn-outperiod.Whiletheonsetofwhitelinediseasecanbeinsidious,thediseasewasmildandthehorserespondedwelltosupportivemanage-ment.Anypossibleimpactonwoundhealingduetounevenweightdistributionwasconsideredunlikely.Additionally,thediseasewasnotapparentduringtheMTtestingperiodandthehorsedidnotdemon-strateanyphysiologicalorbehaviouralidentiﬁersfordiscomfort.Finally,woundhealingassessmentsonday25wereperformedbetweendays23and27dependingonthedayoftheweek.Theimpactondatainterpretationwasconsideredminimal,aseachhorseofferedoneLBAC-treatedandonesaline-treatedwound.CONCLUSIONSThetopicalapplicationofLBACimmediatelyfollow-ingwoundinganditsreapplication24hourslaterincreasedtheaveragemechanicalforcerequiredtoelicitabehaviouralresponseonday1.TheapplicationofLBACondays2and3didnotresultinclin-icallyappreciableantinociception.Additionally,theuseofLBAConcedailyfor7daysdidnotadverselyaffecttherateofwoundhealingoverthe25-daystudyperiod.However,furtherresearchisrequiredtoinvestigatetheantinociceptiveandwound-healingeffectsofLBAConnaturallyoccurringpost-traumaticwoundsinhorses.AUTHORCONTRIBUTIONSAlbertSole-Guitart,JoanneRaingerandWendyGood-wincontributedtoconceptualisation,methodology,investigation,softwareandwriting—reviewandedit-ing.WendyGoodwinadditionallycontributedtodatacuration,supervision,projectadministration,resourcesandfundingacquisition.KarladeKlerkcontributedtotheinvestigation,datacuration,for-malanalysisandwriting—originaldraftpreparation,reviewandediting.ChiaraPalmiericontributedtodatacuration,formalanalysisandwriting—reviewandediting.ShaunPrattcontributedtoformalanalysis,investigation,datacurationandwriting—originaldraftpreparation,reviewandediting.Eliz-abethEvansandJaneHumecontributedtocon-ceptualisation,methodology,projectadministrationandwriting—reviewandediting.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.ACKNOWLEDGEMENTSTheauthorswouldliketoacknowledgethefollowingindividualsfortheirextensivecontributions.Veron-icaSmithfromMedicalEthicsforprovidingproductinformationandstatisticaladvice.MitchCoyleandRebeccaCameronfromtheUniversityofQueenslandSchoolofVeterinaryScience’sEquineUnitfortheirassistancewithanimalmanagement.JoGordonfromtheUniversityofQueenslandSchoolofVeterinaryScience’sPathologyDepartmentfortissuehandlingandpreparation.BiancaAmietfromInvetusforstudymanagement.ThestudywasfundedbyInvetus(grantrecord2018002127).CONFLICTOFINTERESTSTATEMENTThestudywassponsoredbythemanufacturerofthemedication—AnimalEthics.Invetuswasthevet-erinarycontractresearchorganisationthatimple-mentedthestudyinconjunctionwiththeUniver-sityofQueensland(UniversityofQueenslandgrantrecord:Invetus2018002127).AuthorsE.E.andJ.H.wereemployeesofInvetusduringtheexperimentaldesign,conductandanalysisofresults.Thedatainter-pretationandpreparationofthemanuscriptwereassistedbyVeronicaSmith,anemployeeofMedicalEthics.DATAAVAILABILITYSTATEMENTThefulldatasetcanbemadeavailableuponrequesttothecorrespondingauthor.ETHICSSTATEMENTAllprocedureswereperformedwiththeapprovaloftheUniversityofQueensland’sProductionandCompanionAnimalEthicsCommittee(SVS/300/18/INVETUS).Informedconsentwasobtainedfromtheownerortheauthorisedagentfortheowneroftheanimalsenrolled.REFERENCES1.AnantamaNA,DuCheyneC,MartensA,RothSP,BurkJ,DeSpiegelaereW,etal.Thegranulation(t)issue:anarra-tiveandscopingreviewofbasicandclinicalresearchoftheequinedistallimbexuberantwoundhealingdisorder.VetJ.2022;280:105790.2.TextorJA,ClarkKC,WalkerNJ,AristizobalFA,KolA,LeJeuneSS,etal.Allogeneicstemcellsaltergeneexpressionandimprovehealingofdistallimbwoundsinhorses.StemCellsTranslMed.2018;7:98‒108. 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VETERINARYRECORD 13of13 bufferonwoundhealinginratskin.WoundRepairRegen.2011;19:223‒28.SUPPORTINGINFORMATIONAdditionalsupportinginformationcanbefoundonlineintheSupportingInformationsectionattheendofthisarticle. Howtocitethisarticle:PrattS,Sole-GuitartA,deKlerkK,EvansE,HumeJ,PalmieriC,etal.Antinociceptiveandwoundhealingeffectsofacommercialformulationoflidocaine,bupivacaine,adrenalineandcetrimideappliedtopicallytosuperﬁcialskinwoundsinhorses.VetRec.2024;e4395.https://doi.org/10.1002/vetr.4395

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VeterinaryScience,UniversityofQueensland,Gatton,Queensland,Australia.Email:shaun.pratt@uq.edu.auFundinginformationInvetus,Grant/AwardNumber:2018002127 AbstractBackground:Post-traumaticdistallimbwoundscausediscomfortandhealgraduallybysecondintention.ThetopicalapplicationofTri-Solfen(lido-cainehydrochloride,bupivacainehydrochloride,adrenalineacidtartrateandcetrimide[LBAC])produceseffectivepostsurgicalcutaneousanalgesiainlambs,calvesandpiglets;however,itseffectonwoundsinhorsesisunknown.Methods:Theantinociceptiveeffect,measuredbymechanicalthreshold(MT),andthewoundhealingimpactsofLBACcomparedwithsalinewereinvestigatedonsurgicallycreated20×20mmdistallimbwoundsin10horses.Treatmentwasappliedoncedailyfor7daysfollowingwoundingonday0.Mechanicalthresholdsweremeasuredaftertreatmentondays1,2and3.Healingwasobservedfor25days.Results:ThetopicalapplicationofLBACimmediatelyfollowingwoundinganditsreapplication24hourslaterincreasedtheaverageMTontheﬁrstpost-traumaticdayby3Newtons.However,noantinociceptivebeneﬁtwasobservedondays2or3.TreatmentwithLBACdidnotadverselyaffectwoundhealingwhencomparedwithsaline.Limitations:MethodologicaldifferencesprecludeabsoluteMTcomparisonsbetweenstudies.Theexperimentaldesigndidnotincludeamodelofcontaminatedornaturallyoccurringwounds.Conclusion:LBACmayprovideanearlyantinociceptivebeneﬁtwhenappliedtouncontaminatedsurgicallycreatedwoundswithoutcompromisingheal-ing.KEYWORDSantinociception,mechanicalthresholdtesting,Tri-Solfen,woundhealing,horseINTRODUCTIONTraumaticinjurytothedistalextremitiesofhorsesiscommon.Post-traumaticdistallimbwoundscausediscomfort,arenotoriouslydifﬁculttoman-ageandoftenexhibitprotractedsecond-intentionhealing.Thereisconsiderableinterestindevelop-ingnovelapproachestoimprovethehealingrateoflowerlimbwoundsinhorses,1with,histori-

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otractedsecond-intentionhealing.Thereisconsiderableinterestindevelop-ingnovelapproachestoimprovethehealingrateoflowerlimbwoundsinhorses,1with,histori- ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution-NonCommercial-NoDerivsLicense,whichpermitsuseanddistributioninanymedium,providedtheoriginalworkisproperlycited,theuseisnon-commercialandnomodiﬁcationsoradaptationsaremade.©2024TheAuthors.VeterinaryRecordpublishedbyJohnWiley&SonsLtdonbehalfofBritishVeterinaryAssociation.cally,astrongfocusonoptimisingthewoundbedmicroclimate.2Theeffectoftreatmenthasbeeninves-tigatedinexperimentalmodelsofsurgicallycreatedwounds2,3andinclinicaltrialsofnaturallyoccur-ringwounds4;however,suchstudieshavefocusedheavilyonwoundhealingwithoutconsideringanalgesia.Providingrelieffromdiscomfortisthecorner-stoneoftraumamanagement.Cutaneouswounds VetRec.2024;e4395.wileyonlinelibrary.com/journal/vetr 1of13https://doi.org/10.1002/vetr.4395

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2of13 VETERINARYRECORD

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2of13 VETERINARYRECORD causediscomfort,reduceperformance,compro-misethehuman‒animalbondandmayresultinlocalisedhypersensitisationwithassociatedbehaviouralavoidance.5Althoughnon-steroidalanti-inﬂammatoriesareeffectivesystemicanalgesicsforthetreatmentofwound-associatedpaininhorses,clinicallyvalidatedtopicaltherapiesremainscarce.Thisisimportant,as60%ofhorseownersapplypotentiallycytotoxiccompoundstowoundspriortoveterinaryconsultation.6Thisrisksdisruptiontoearlyhaemostaticandﬁbroproliferativecellsandmayreducetheeffectivenessofprospectivetherapiesprescribedbyveterinaryclinicians.Itis,therefore,importanttoprovideownerswithalicencedproductthat(1)deliverseffectiveanalgesiaand(2)preserveswoundhealingpotential.Tri-Solfen(AnimalEthics)isanaqueousﬁxedcombinationproductcontaining40.6g/Llidocainehydrochloride,4.2g/Lbupivacainehydrochloride,24.8mg/Ladrenalineacidtartrateand5.0g/Lcetrim-ide(LBAC).Theproductlabelclaimstoproviderapidanalgesia,visiblyreducebloodlossandimprovewoundhealingwhenappliedtopicallytocutaneouswounds.TheLBACformulationhas36patentsgrantedglobally,includingintheUnitedStates,Europe,NewZealandandGreatBritain(SmithV,MedicalEthics,personalcommunication,2023).InAustralia,LBACisregisteredfortheprovisionofpostoperativeanalge-siafollowinginvasivesurgicalproceduresinlambs,7–9calves10andpiglets.11,12Priortothisstudy,theeffectsofLBAConequinewoundswereunknown.Thisstudywasdesignedasapharmaceuticaltrialtocomparetheantinociceptiveeffects,measuredbymechanicalthreshold(MT)testing,oftopicallyappliedLBACcomparedwith0.9%sodiumchlorideonsurgicallycreateddistallimbwoundsinhorses.TheinﬂuenceofLBACtreatmentonwoundhealingwasasecondaryaimtoensuretheabsenceofadverseeffects.ItwashypothesisedthatLBACwouldprovideshort-durationantinociception(increasedMTs)with-outadverselyaffectingwoundhealing.TheresultsofthisstudyprovidedtheevidencethatledtotheLBACformulation,marketedasTri-Solfen,receiv-ingregistrationwiththeAustralianTherapeuticGoodAssociationasanon-prescriptionveterinaryproductforuseonpost-traumaticequinewounds.MATERIALSANDMETHODSAnimalsAllprocedureswereperformedwiththeapprovaloftheUniversityofQueensland’sProductionandCompanionAnimalEthicsCommittee(SVS/300/18/INVETUS).Tenhorses(ninegeldingsandonemare)aged7.3±3years,belongingtotheUniversityofQueensland,wereenrolledinthestudy.ThepopulationincludedfourStandardbreds,oneStandardbredcrossandﬁveThoroughbreds.Allani-malswereconsideredhealthybasedonaphysicalexaminationandmetthefollowinginclusioncrite-ria:morethan2yearsofage,indateHendravirusandtetanusvaccination,easyandsafetohandle,nolamenessoridentiﬁableskinlesionspresent,7dayswithheldfromallmedications(includingnon-steroidalanti-inﬂammatories)andnoknownpreviousadversereactionstolocalanaestheticsorinjectablesedatives.Atthecompletionofthestudy,allani-malswerereturnedtotheUniversityofQueensland’steachingandresearchherd.SummaryofstudydesignThestudywasaprospective,randomised,blinded,longitudinalcontrolledstudy.ThestudywasdesignedandexecutedaspertheCONSORTguidelines.13Fol-lowingthecreationofbilateralsurgicalwoundsonthethirdmetacarpi,eachlimb(leftorright)wasran-domisedusingacointosstoreceivetopicaltreatmentwitheitherLBACor0.9%sodiumchloride(saline).Theantinociceptiveandwoundhealingeffectswereassessedbyveterinarianswhowereblindedtothetreatmentallocation.Afulldescriptionofthemechan-icalstimulusisprovidedbelow;however,earlierworkinhorses,usinganidenticalcontrolunitandpneu-maticactuatorconﬁguration,deﬁnedtheMTofintactepidermisas3.1±2Newtons(N).14Todetect3NofdifferenceinMTsbetweentreatments,atotalofeightwoundswerethereforerequiredpertreatmentgroup(α=0.05andβ=0.8).TreatmentTheLBACtreatmentcontained40.6g/Llidocainehydrochloride,4.2g/Lbupivacainehydrochloride,24.8mg/Ladrenalineacidtartrateand5.0g/Lcetrim-ideinaﬁxedcombinationsolutionthatwasblueincolour.Tomaintainblindingtotherelevantstudypersonnel,thesalinecontrolsolutionwassimilarlycolouredbyadding2.5mLofbluedye(QueenBlueFoodColour)containing1.8%totaldyestuffto250mLof0.9%sodiumchloride(BaxterHealthcare).Identi-calspraybottleswereusedtoapply1mLtopically(2×0.5mLspray)ofeachsolutiondirectlyontotheallocatedwoundarea(1mLper20mm2).Thiswassufﬁcienttocovertheentireareawithminimalrunoff.Bothsolutionswerestoredinaninsulatedboxinatemperature-controlledroom(<25 C)whennotinuse.MechanicalthresholdtestingAwirelessMTtestingsystem(WMT2LargeAni-mal,TopcatMetrology)wasusedforthestudy.TheapplicationofthedevicewasidenticaltopreviousdescriptionsbyTaylor.15Brieﬂy,themechanicalstim-uluswassuppliedbypneumaticactuators,whichdroveanearlyhemispherical1mmpinattachedto

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VETERINARYRECORD 3of13

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VETERINARYRECORD 3of13 FIGURE1(a)Thecontrolunitandreservoirdevicemountedoverthehorse’swithersandsecuredwithVelcrotoalightweightbellystrap.Pressuretubingconnectsthecontrolandreservoirdevicetothepneumaticactuatorﬁttedtoeachmetacarpus.(b)Thepneumaticactuatorsﬁttedtoeachmetacarpusandsecuredwithmodiﬁedbrushingbootsamovingpistonmountedonarollingdiaphragm.Thepneumaticactuatorswereﬁttedtotheanterioraspectofeachmetacarpusandwereheldinplacebymodiﬁedbrushingboots.Thecontrolunitwasmountedoverthehorse’swithersandsecuredwithVelcrotoalightweightbellystrap.Arechargeablepres-surereservoir(60mLsyringe)wasmountedalongsidethecontrolunitandwasconnectedviapressuretub-ingtothepneumaticactuatorunits(Figure1).AsinglemodiﬁcationtotheMTdeviceinvolvedtheadditionofasmallsiliconcoveringatopthe1mmpin.Thisapproachaimedtopreventlocalisedtissuetraumaassociatedwithrepeattesting.Thesiliconcoveringﬁt-tedtightlyandfollowedtheroundedcontourofthepinhead.Thecontrolunitsequentiallyincreasedtheforcegeneratedbytheactuatorsthroughthe1mmpinatarateincreaseof0.8N/second,from0to20N.Thetest-inglimitwassetat20N.TheoverallMTforeachlegwasrecordedasthemeanofatleasttwotestsrecordedwithin2Nofeachother.Thetestingintervalwas5minutes.Bothactuatorswereﬁtted;however,onlyonelegwastestedatatime.Apositiveresponse(indicatingMT)wasdeﬁnedastheliftingofalegorfoot,pawingattheground,stampingafoot,ﬂexingtheantebrachiumorwalkingtoavoidstimulus.16AtallMTcollectionpoints,theoperatorremainedoutsidethestallandthehorsewasfreetomoveaboutitsstall.Testingbeganwheneachhorsewasvisiblyrelaxedandnotdistractedbyenvironmentalstim-uli.Insectrepellent(N-diethyltoluamide,di-N-propylisocinchomeronate,N-octylbiccloheptenedicarobox-imide,piperonylbutoxide,citronella,pyrethrinsolu-tion;Repel-XTroyLaboratories)wasusedtominimiseconfoundingbehaviourduringMTtesting.TolimitthelearningeffectassociatedwithrepeatMTtest-ing,alleffortsweremadetominimisehorseroutineandnotestingcueswereused.Additionally,irrespec-tiveoftreatmentallocation,odd-numberedhorsesunderwentMTtestingoftheleftlegﬁrst,whileeven-numberedhorsesunderwenttestingoftherightlegﬁrst.ExperimentalprotocolAﬂowdiagramdepictingtheexperimentalproto-colisshowninFigure2.Inthe7dayspriortotheexperimentalphase,eachhorsewasweighed,assignedanumberedtagandunderwentafullclini-calandlamenessexamination.Animalsenteringtheexperimentalphase(startingfromday−1)wereran-domised,groupedandstaggered7daysapart.Horsesweresequentiallynumberedastheyenteredthestudy(horses2‒11).Horse1wasapilotanimalthatdidnotcontributetothestatisticalanalysis.Day–1:acclimatisationPairedhorseswerebroughtinfromtheirpaddocksandhousedinshelteredstallsmeasuring5×5m.Foracclimatisationpurposes,eachhorsewasﬁttedwiththeMTdeviceinanidenticalfashiontohowitwouldbeﬁttedfortesting.Thedevicewasremovedafter20minutes.NoMTtestingwasperformed.Day0:baselineMTtesting,surgicalwoundingandtreatmentapplicationBaselineMTtestingonintactepidermiswasper-formedpriortosurgicalwoundingwiththedeviceﬁttedaspreviouslydescribed.Toreducecross-contaminationbetweenhorses,thetestingsitewascoveredwithanantimicrobialincisedrape(3MIoban2AntimicrobialInciseDrape6640EZ,3M).FollowingbaselineMTdatacollection,horseswererestrainedinstockswithcrosstiesandsedatedwith0.01mg/kgintravenous(IV)detomidine

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4of13 VETERINARYRECORD FIGURE2Timelineoftheexperimentalprotocol.Animalsenteringtheexperimentalphase(startingfromday−1)wererandomised,groupedandstaggeredsevendaysapart.Mechanicalthreshold(MT)datawerecollectedinstalls,whilewounding,treatmentapplicationandwoundassessmentwereperformedinstocks

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dandstaggeredsevendaysapart.Mechanicalthreshold(MT)datawerecollectedinstalls,whilewounding,treatmentapplicationandwoundassessmentwereperformedinstocks FIGURE3Anexampleofsurgicallycreatedbilateralthoraciclimbwoundsonthedorsal45 medialaspectofthemid-thirdmetacarpi.Theimageisofhorse8onday0hydrochloride(EqusedanVet,Ausrichter).Fivemin-uteslater,thesurgicalsiteonboththoraciclimbswasclippedandpreparedusingacombinationof5%chlorhexidinescruband70%alcoholspray.Aringblockusing10mLof2%lidocaine(Lignocaine20,TroyLaboratories)wasperformedproximallytothesurgicalarea.Asterile,autoclavedstencilandrulerwereusedtoreproducesurgicalwoundsinallhorses.Afull-thickness20×20mmsurgi-calwoundwascreatedonthedorsal45 medialaspectofthemid-thirdmetacarpiusinganumber10scalpelblade(Figure3).Thesurgicalsectionswereﬁxedin10%neutralbufferedformalinwithatissue-to-formalinratioof1:10ormore.Toreduceexcessivehaemorrhage,thewoundswereinitiallycov-eredwitha5×5cmlowadherent,sterileabsorbentdressing(Cutilin,Smith&Nephew).After2hours,theabsorbentdressingwasremovedandthetreat-ment(LBACorsaline)wasappliedtotheallocatedwound.Once30minuteshadelapsed,thetreatedwoundswerebandagedwithanabsorbentdressingandalayerofadhesivebandage(AskinaPlastE,BBraun)andcoveredwithcommerciallyavailablesta-blewraps.Horseswerereturnedtotheirstalls.Nofurtherexperimentalprocedureswereperformedonday0.Days1‒3:antinociceptiveMTtestingpriortoandfollowingtreatmentapplicationThestablewrapsanddressingswereremovedeachday,andanantimicrobialincisedrapecuttosuf-ﬁcientlycoverthewoundwasappliedtoprotectthetissuefromtheMTdeviceandtominimisecross-contaminationbetweenwounds.PretreatmentMTtestingwasperformedwithhorsesinstalls,asdescribedpreviously.Theantimicrobialincisedrapewasthenremoved,andhorseswererestrainedinstocksfortreatmentreapplication.EachhorsewastreatedwithLBACorsalineontheallocatedwoundatthesametimeeachday(±15minutes)bythesametwounblindedstudypersonnel.Thetreatedwoundswereleftuncoveredfor20minutesbeforeanewantimi-crobialincisedrapewasapplied.Post-treatmentMTtestingwasperformedwithhorsesfreeinstallsat0.5,1and3.5hoursfollowingtreatmentapplication.Theactuatorpinofthepneumaticdevicewithitssiliconcoveringwascentredwithinthedorsolateralquadrantofthewoundedarea.Oncetestingwascom-plete,theantimicrobialincisedrapewasremovedandthewoundswererebandagedaspreviouslydescribed.

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VETERINARYRECORD 5of13 Days4‒6:treatmentapplicationonlyTreatmentcontinueddaily.NoMTtestingwasper-formed.Days7‒25:turnoutOnday7,alldressingswereremoved,andthehorseswereindividuallyturnedoutintoa12×6mgrassypaddockuntilday25.Day25:histopathologysamplingOnday25,thehorseswereagainrestrainedinstocksandsedatedwith0.01mg/kgIVdetomidinehydrochloride.Once5minuteshadelapsed,theskinwassurgicallypreparedandaringblockwasper-formedusinglidocaineaspreviouslydescribed.An8mmbiopsypunchwastakenfromthedorsome-dialwoundmarginandﬁxedin10%neutral-bufferedformalin.Histopathologicalanalysisofthepairedsamples(days0and25)wasperformedbyablindedveteri-narypathologist.Tissuesampleswereembeddedinparafﬁn,sectionedatathicknessof5µmandstainedwithhaematoxylinandeosinandcollagen-speciﬁcMasson’strichromestaining.ThehistologicalcriteriaforwoundhealingwereextrapolatedfromTheoretetal.17Brieﬂy,sectionsoftissuewereevaluatedforthepresenceorabsenceofsurfaceulceration,gran-ulationtissue,keloidalcollagen,haphazardcollagenﬁbrearchitecture,ﬁbroblastinﬁltrationandﬁbrinoidnecrosisofthedermalvessels.Thedegreesofder-malvascularity,inﬂammation,ﬁbrosis,haemorrhage,inﬁltrationofeosinophilsandﬁbroblastcellularityweregradedcategoricallyfrommildtosevere.Finally,acomparativestageofhealingatday25wasassessedbetweentreatments.WoundassessmentWoundswerephotographedondays0,4,11,18and25.Thewoundareawasmeasuredusingimageanaly-sissoftware(ImageJ,USNationalInstitutesofHealth)witha60×60mmrulerheldadjacentforstandardref-erence(Figure4).Theareaofeachwoundinmm2wasmeasuredthreetimesandtheaveragewascalculatedtomakeasinglemeasurementforeachday.Thiswasusedtocalculatethewoundarea(mm2)andtherateofhealingdependentontheelapsedday.Rateofhealingdependantonelapsedday=−1 woundarea2−woundarea1

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eachday.Thiswasusedtocalculatethewoundarea(mm2)andtherateofhealingdependentontheelapsedday.Rateofhealingdependantonelapsedday=−1 woundarea2−woundarea1 time2−time1 mm2 day Additionally,thephotographswereusedtogradetheamountofmacroscopicgranulationtissuepresentondays4,11,18and25accordingtothefollowing FIGURE4Anexampleofwoundassessment(horse8onday18).A60×60mmtemplatewasusedforstandardreferenceduringphotographicassessmentofwoundhealing.Imagesweremeasuredusingimageanalysissoftware(ImageJ)standards:gradeI,depressedbelowtheskinedge;gradeII,proliferatedtotheleveloftheskinedge;gradeIII,elevatedabovetheskinedge;andgradeIV,requiredtrimmingbacktotheleveloftheepitheliumtocontrolexcessiveproliferationthatinhibitedtheadvancementoftheepithelium.GradeIVgranulationtissuewasconsideredexuberant.18Allmacroscopicgradingswereperformedbythesameblindedindi-vidualinrandomsuccessionatthecompletionofthestudy.GeneralhorsewelfareFreshwaterwasprovidedadlibitumandlucerneorgrassyhaywasprovidedtwicedailythroughoutthestudy.Afterday7,horsesalsohadaccesstograss.Dailyexaminationsrecordedthedemeanour,eating,drinking,urinating,defecationandgeneralbehaviourofallanimalsfromday−1today25.Ondays0‒3,allhorsesunderwentaphysicalexaminationandwerepainscoredbyaveterinarianblindedtotreatmentapplicationusingavisualanaloguescale(VAS).TheVASwasa100mmlinewith0representingnopainand100representingimmensepain.IfanyhorsescoredaVASof40mmormore,rescueanalgesia(methadone0.1mg/kg)andsedation(acepromazine0.01mg/kg)wereadministeredIV.Horseswerereassessedafter30minutesandadministeredﬂunixinmeglumine1.1mg/kgIViftheVASscoreremained40mmorgreater.Horsesthatreceivedrescueanalgesiawereexcludedfromthestudy.StatisticalanalysisThedatawerecomparedusingeitherGenStat(Release22.1,VSNInternational)orTIBCOSpotﬁreS+8.2

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6of13 VETERINARYRECORD

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6of13 VETERINARYRECORD (2010).Descriptivesummariesarepresentedasthemean±standarderrorofthemean.Mechanicalthresholdmeanswerecomparedusingalinearmodelwiththefollowingﬁxedvariables:treatment(LBAC,saline),day(1,2,3),timepointwithinday(0.5,1,3.5hours),replicateandtestleg(leftorright).Themodel’ssuitabilitywasassessedusingplotsofresiduals.Addi-tionally,MTdatawerecomparedwithineachdayusingthesamemodel(withdayremoved)andsuit-abilityassessment.Signiﬁcancewassetatap-valueoflessthan0.01.Woundhealingdata,includingwoundareaovertimeandwoundhealingrate,werecomparedusingalinearmodelwiththefollowingﬁxedvariables:treatment(LBAC,saline),day(0,4,11,18,25)andtestleg(leftorright).Themodel’ssuitabilitywasassessedusingplotsofresiduals.Additionally,woundareaandwoundhealingratewerecomparedwithineachdayusingthesamemodel(withdayremoved)andsuit-abilityassessment.Signiﬁcancewassetatap-valueoflessthan0.05.RESULTSNoVASscorewasabove0foranyhorse,andnores-cueanalgesiaorsedationwasprovidedtoanyhorse.Onehorsedevelopedwhitelinediseaseintherightthoracicfootonday13(LBAC-treatedlimb).Thehorsewassoundatthetrotandnegativetohooftesters.Thehorsewastreatedwithcorrectiveshoeingandapoultice.ThisanimalwasnotexcludedfromthestudybecauseMTtestinghadalreadyconcluded.Onehorse(horse3)waswithdrawnfromthestudyonday1duetoabnormalitiesonphysicalexamination(tachyp-noeaandventraloedema).Thehorsewasdiagnosedwithachronicdiseaseunrelatedtothestudyanddidnotcontributetostatisticalanalysis(n=9).MechanicalthresholdtestingTwotothreereplicateMTmeasurementsweresuf-ﬁcienttogeneratereproduciblereadings(<2N)forallanimals.TheaveragemechanicalforcerequiredtoelicitabehaviouralresponsepriortowoundingwassimilarbetweentheLBAC(4.2±0.6N)andsaline(4.2±0.5N)groups.TheaverageMTmeasuredatbaselineandfollowingtreatmentreapplicationondays1,2and3isillustratedinFigure5.ThelinearpredictivemodelfortheMTdataisshowninTable1.Overall,treatmentwithLBACincreasedtheaverageMTrequiredtogenerateabehaviouralresponsewhencomparedwithsaline(p<0.001).Themostsigniﬁcanttreatmenteffectwasseenonday1(p=0.003).Onday1,theaver-ageincreaseinMTfollowingtreatmentwithLBACwas3.0N(minimum1.0Nandmaximum5.0N).Thiseffectwasnotinﬂuencedbytime(timepointp=0.441),indicatingthat,comparedwithsalinetreatment,LBACtreatmentsigniﬁcantlyincreasedtheaverageMTatalltimepoints(0.5,1and3.5hours)onday1(Figure5).Onday2,treatmentwithLBACdidnotaltertheMTcomparedwithsaline(p=0.131).Onday3,theaverageincreaseinMTfollowingtreatmentwithLBACcomparedwithsalinewas2.3N(minimum0.5Nandmaximum4.0N)(p=0.011);however,thiseffectwassigniﬁcantlyinﬂuencedbytime(timepointp=0.004)andfellbelowthepowerofdetectionandstatisticalsigniﬁcanceforthestudy.WoundhealingLimbwoundsrandomisedtoreceivetreatmentwithLBACwereinadvertentlycreatedlargerthanthoseran-domisedtoreceivetreatmentwithsaline(LBAC:360±12mm2andsaline:342±10mm2).Theareaofallwoundsincreasedonday4beforereducingondays11,18and25(Figure6).Comparablehealingrateswerefoundbetweentreatmentsoverthestudyperiod(Figure7).Thelinearpredictivemodelfoundnodifferencebetweentreatmentswithrespecttohealingrate(p=0.717)orwoundarea(day0,p=0.281;day4,p=0.555;day11,p=0.244;day18,p=0.119;day25,p=0.131).HistopathologyandgrosstissueassessmentForbothtreatmentgroups,thesurgicalsectionscol-lectedonday0wererepresentativeofhealthytissue.Histopathologicalanalysisofbiopsysamplescol-lectedonday25revealedthatfourwoundstreatedwithLBACwereslightlyimmaturewhencomparedwiththoseofthecontralaterallimbtreatedwithsaline,twowoundstreatedwithsalinewereslightlyimmaturewhencomparedwiththecontralaterallimbtreatedwithLBACandthreewoundswereincomparablestagesofhealing(Table2).Overall,allwoundsinbothtreatmentgroupsexhibitedheal-ingprogressionthatwasappropriateforthestudydurationandwoundtype.Thecompletehistopatho-logicalgradeforeachwoundisavailableinthesupportinginformation.Noexuberantgranulationtissue(EGT)formedwithinanywoundtreatedwithLBACorsaline(Table3).Allwoundsexhibitedgranulationtissuebelowtheskinedgesonday4.Ondays11,18and25,sixLBAC-treatedandsixsaline-treatedwoundswerescoredasgradeIII(Figure4)and16LBAC-treatedand14saline-treatedwoundswerescoredasgradeII.DISCUSSIONThisstudyusedMTtestingtoinvestigatetheantinoci-ceptivepropertiesofacommercialformulationofLBACappliedtopicallytosurgicallycreatedfull-thicknesswoundsonthedistalthoraciclimbsofhorses.TreatmentwithLBACfollowingwoundinganditsreapplication24hourslaterincreasedtheaver-agemechanicalforcerequiredtoelicitabehaviouralresponse.Thepeakantinociceptiveeffectappearedto

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VETERINARYRECORD 7of13 FIGURE5Averagemechanicalthresholds(MT)measuredfromninehorsestreatedwitheitherlidocaine‒bupivacaine‒adrenaline‒cetrimide(LBAC)orsalineappliedtopicallytosurgicallycreateddistalthoraciclimbwounds.MTwasmeasuredonday0priortowounding(baseline)andoneachconsecutivedaypriortotreatment(0)andat0.5,1and3.5hoursfollowingtreatment.Thedataareexpressedasthemean±standarderrorofthemean.Asteriskindicatesstatisticalsigniﬁcanceextrapolatedfromthelinearpredictivemodel(p<0.01)TABLE1Treatmentwithlidocaine-bupivacaine-adrenaline-cetrimide(LBAC)orsalineappliedtopicallytosurgicallycreateddistalthoraciclimbwoundsinninehorses.Averagemechanicalthresholdsbetweentreatmentswerecomparedusingaﬁxedeffectslinearmodel. ParameterFstatisticp-ValueLower95%CI(N)Pointestimateofdifference(N)Upper95%CI(N) OverallTreatment17.78<0.0011.22.23.3Day4.490.012Timepoint2.270.079Replicate0.020.880Testleg0.160.685Day1Treatment8.930.0031.03.05.0Timepoint0.900.441Replicate0.010.918Testleg0.240.626Day2Treatment2.310.13101.43.2Timepoint0.440.725Replicate0.010.929Testleg1.070.303Day3Treatment6.690.0110.52.34.0Timepoint4.650.004Replicate0.090.768Testleg0.050.828

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.310.13101.43.2Timepoint0.440.725Replicate0.010.929Testleg1.070.303Day3Treatment6.690.0110.52.34.0Timepoint4.650.004Replicate0.090.768Testleg0.050.828 Abbreviation:CI,conﬁdenceinterval.Boldingwasusedtohighlightthestatisticallysigniﬁcantvaluesinthetable.occur0.5‒1hourafterthereapplicationofLBAConday1(24hoursafterwoundcreation).Noapprecia-bleantinociceptivebeneﬁtwasdetectedfollowingthereapplicationofLBAConday2or3(48and72hoursafterwoundcreation).Additionally,theuseofLBACappliedtopicallyoncedailyfor7dayshadnoadverseeffectsonwoundhealingoverthe25-daystudyperiod.NociceptivethresholdtestingNociceptivethresholdtesting(NTT)isarecognisedanalgesiometrictechniqueusedtoinvestigatetheefﬁcacyofanintervention.5Itinvolvesapplyinganox-iousstimulus(mechanical,electricalorthermal)inanintensifyingmanneruntilthedesiredbehavioural

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8of13 VETERINARYRECORD FIGURE6Changesinwoundareainninehorsestreatedwitheitherlidocaine‒bupivacaine‒adrenaline‒cetrimide(LBAC)orsalineappliedtopicallytosurgicallycreateddistalthoraciclimbwounds.Measurementsweremadeusingimageanalysissoftware(ImageJ)andareexpressedasthemean±standarderrorofthemean

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callytosurgicallycreateddistalthoraciclimbwounds.Measurementsweremadeusingimageanalysissoftware(ImageJ)andareexpressedasthemean±standarderrorofthemean FIGURE7Woundhealingrateforninehorsestreatedwitheitherlidocaine‒bupivacaine‒adrenaline‒cetrimide(LBAC)orsalineappliedtopicallytosurgicallycreateddistalthoraciclimbwounds.Measurementsweremadeusingimageanalysissoftware(ImageJ)andareexpressedasthemean±standarderrorofthemeanresponseiselicited.16Thedifferenceinnociceptorthresholdspriortoandfollowingtheapplicationoftheinterventioninfersitsantinociceptiveeffect.MTtestingisaneffectivemethodtoquantifythenocicep-tivethresholdinnon-verbalanimals.19Whenusedasanon-invasivenociceptivechallengetostudywound-associatedpaininhorses,MTtestingwasmoresen-sitiveandspeciﬁcthanelectricalorthermaltesting.14Comparedwiththermalthresholdtesting,MTtest-inggeneratesareproducibleforcestimulus,canberemotelycontrolledandcarrieslesslocalisedtissuetrauma.14However,therearelimitationstoMTtest-ing.Theseincludeconditioningbiasandrepeattestingtrauma.20,21Withrepeattesting,horsesmayassociatetheliftingofalegwiththereleaseofpressureandmayproceedtolifttheirlegatprogressivelylowerthresholds.Thisresultsinatemporalshiftinthethresholddataandistermedlearningbias.Similarly,horsesmayassociatethetestingapparatusortestingcueswithimpendingnoxiousstimuli.20,21TheadvantagesofthewirelessMTdevicewereitslightweightattachmentsanditsportablehandheldtransmitter.Thisallowedallper-sonneltoremainoutsidethestallduringtestingandminimisedtheamountofequipmentrequired.Testingcueswereomitted,andtheorderoflimbstestedwasrandomised.ThereareimportantconsiderationswithinthemethodsthatneedtobeaddressedinrelationtotheMTresults.First,toreducetheincidenceofrepeattestingtrauma,asiliconecoveringwasplacedoverthe1mmpin,whichwasdrivenbythepneumaticactuators,duringallMTdatacollectionpoints,includ-ingbaseline.Topreventwoundsitecontaminationandcross-contaminationbetweenhorses,allwoundswerecoveredwithathinantimicrobialincisedrapeduringtesting.TheseadjustmentslikelyaccountedfortheonaveragegreaterMTrecordedpriortowounding(intactepidermis)comparedwithaprevi-ousreport.14Intranslationalhumanstudies,probesofsmallercross-sectionalareareducescatterwithinMTtesting,22suggestingthattheadditionofasili-concapmayalsoexplainthevariabilityinthepresentdata.TheonaveragegreaterMTonintactepider-mismaycausethepowercalculationtounderesti-matetherequirednumberofwounds.Toaccountforthescatterandpotentiallyreducedstatisticalpower,thep-valueforMTsigniﬁcancewassettolessthan0.01.Finally,itisimportanttoconsiderthedifferencebetweenpressureandforce.Pressureequatestotheforceappliedperunitarea.Anincreaseinsurfaceareabytheadditionofthesiliconcapmayreducepres-suretransfertoindividualperilesionalnociceptors,yetmayincreasethetotalnumberofperilesionalnocicep-torsstimulated.Thenon-linearrelationshipbetweenprobediameterandresultsfromMTtestingisshownbyTayloretal.23Thismodiﬁcationpreventsthedirectcomparisonofresultswithotherstudies;however,theauthorsbelievethattheconservedmethodology

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VETERINARYRECORD 9of13 TABLE2Summarisedhistopathologicalanalysisofsurgicallycreateddistalthoraciclimbwoundsinninehorsestreatedtopicallywitheitheracommercialformulationoflidocaine‒bupivacaine‒adrenaline‒cetrimide(LBAC)orsaline LBACSaline Day0Day25Day0Day25(n) DermalvascularityNoincreaseorminimal9090Mild0000Moderate0706Severe0203DermalinﬂammationAcute08a07Chronic02a02DermalinﬂammationAbsent9190Minimal0000Mild0202Moderate0403Severe0204DermalﬁbrosisAbsent9090Minimal0000Mild0302Moderate0607Severe0000FibroblastcellularityNoincreaseorminimal9090Mild0000Moderate0807Severe0102DermalhaemorrhageAbsent9090Mild0307Moderate/severe0602InﬁltrationofeosinophilsintothedeepdermisAbsent9090Minimal0104Mild0605Moderate/severe0200SurfaceulcerationAbsent9191Present0808GranulationtissueAbsent9090Present0909KeloidalcollagenAbsent9999Present0000HaphazardcollagenﬁbrearchitectureAbsent9897Present0102 (Continues)TABLE2(Continued) LBACSaline Day0Day25Day0Day25(n) FibroblastsAbsent0000Present9999FibrinoidnecrosisofthedermalvesselsAbsent9798Present0201

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(Continues)TABLE2(Continued) LBACSaline Day0Day25Day0Day25(n) FibroblastsAbsent0000Present9999FibrinoidnecrosisofthedermalvesselsAbsent9798Present0201 Note:Valuesrepresentthenumber(s)ofhorsesﬁttingeachcategory,withthehighestpossiblenumberbeinganine.aOnewoundtreatedwithLBACdisplayedindicatorsforacuteandchronichaemorrhageonday25.remainssufﬁcienttoconcludeontherelativeeffectofLBACwithinthemodeldescribed.NoxiousthresholdtestingbyMThasbeenval-idatedonlyonintactepidermis.Previousstud-ieshaveappliedmechanicalstimulustotheaxialmusculature,24,25face,26regionsofthefoot27,28andmetacarpus29–32toinvestigatetheantinociceptivepropertiesofvariouscompoundsinhorses.TheextrapolationofMTdatacollectedfromintactder-misinsuchstudiesiscautionedduetodifferencesinpatientsensitisationandtheremovalofdermalnoci-ceptorsfollowingwounding.Full-thicknesswoundsareexpectedtoremovenociceptorterminalswithinthedermisandexposetheunderlyingfatandextracel-lularmatrixoftheinterstitium.Therefore,stimulationofnociceptorslocatedwithintheperilesionalder-misisresponsibleforthetransmissionofnociceptionincutaneouswoundmodels.Shalloworpartial-thicknesswoundsmaybemorepainfulthanwoundsthatpenetratethedeepermusculatureduetothehighdensityofnociceptorswithinthesuperﬁcialdermis.Forexample,thelikelihoodofdevelopingmoderatetoseverewound-associatedpaininhumansis2.7timesgreaterifthewoundispartiallythick.33Consequently,otherformsofNTTthataremorespeciﬁcintheirsomatosensorylocalisationtothewoundperipherywouldlikelygeneratethresholdsofdifferingmagni-tudes.VariableMTsmayalsobeexpectedifthesamedevicewastestedonpartial-thicknesswounds.TheeffectofLBAContheMTwasnotinvesti-gatedonthedayofwoundingasitwashypothesisedthattheeffectoftreatmentmaybedifﬁculttosep-aratefromresidualanalgesiaprovidedforstandingsedation.FailuretotestonthedayofwoundingmayhavepretermittedthetruepeakantinociceptiveeffectofLBAC.ThemarginalincreaseinMTmeasuredonday3mayrepresentmeasurementdriftinthedevice,atype1statisticalerror,localtissuesensitisation21orsimplyachangeindermatomesensitivitywithinthegranulatingtissuemargin.Partitioningthephasesofwoundhealingintodiscretetemporalcategories(haemostasis,inﬂammatory,proliferativeandremod-ellingphase)isanoversimpliﬁcationofthecomplexanddynamicbiologicalprocessesresponsibleforwoundclosure;however,earlyﬁbroproliferationmay

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10of13 VETERINARYRECORD TABLE3Macroscopicgranulationtissuerecordedondays4,11,18and25inninehorsesfollowingtopicaltreatmentofsurgicallycreateddistalthoraciclimbwoundswitheitheracommercialformulationoflidocaine‒bupivacaine‒adrenaline‒cetrimide(LBAC)orsaline LBACSaline Day4Day11Day18Day25Day4Day11Day18Day25 GradeI92119313GradeII06560464GradeIII01320222GradeIV00000000

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aine‒adrenaline‒cetrimide(LBAC)orsaline LBACSaline Day4Day11Day18Day25Day4Day11Day18Day25 GradeI92119313GradeII06560464GradeIII01320222GradeIV00000000 Note:Valuesrepresentthenumber(s)ofhorsesﬁttingeachcategory,withthehighestpossiblenumberbeinganine.havecontributedtothedeclineinantinociceptiveeffectbyday2.AmajorchallengetoNTTisthevariabilityinbehaviouralresponsestoidenticalnoxiousstimulibetweenanimals.Painisanunpleasantsensoryandemotionalexperienceassociatedwithactual,potentialorperceivedtissuedamage.Paininformationcon-nectsviacomplexneurobiologicalpathwaystoaltertheanimal’saffectivestate(behaviour).Forethicalrea-sons,allhorsesreceivedmultimodalanalgesiaatthetimeofsurgicalwounding,includingsubcutaneouslidocainehydrochlorideprovidedasaringblockandtheIVadministrationofdetomidinehydrochloride.Thepre-emptivemodulationofnociceptionintheseanimalstoachievestandingsedationmaynotappro-priatelymodelacutelytraumaticwoundscenarios.TheresultsfromthecurrentstudyindicatethatLBACmaybeeffectiveatincreasingtheMTofsurgicallycre-atedfull-thicknesscutaneouswounds;however,vari-ableresponsesmayoccurinpatientswithtraumaticwoundsorchronicallyinﬂamedwounds.NoxiousthresholdtestingwastheprimaryoutcomemeasureusedtoinvestigatetheantinociceptiveeffectsofLBAConfull-thicknesscutaneouswounds.TheVASwasusedtoassessoverallhorsewellbeingandtodeterminewhethersystemicanalgesicinterventionwasrequiredtotreatpain.Inarecentinvestigation,theVASdisplayedgoodtoverygoodrepeatability,goodtoverygoodreproducibilityandmoderatetogoodvaliditycomparedwiththemultidimensionalCom-positeOrthopaedicPainScale,whenpainwasgradedbyaveterinarian.34TheoverallsensitivityoftheVAStodetectpaininhorsesfollowingundisclosedproce-dureswas96%.34Unfortunately,avalidatedpainscale(‘goldstandard’)iscurrentlyunavailableinhorses.Toensurethathorsewelfareisprioritised,additionalmultidimensionalpainscalesshouldbeconsideredinfuturestudies.WoundhealingComparedwiththoseoftrunklesions,woundsonthedistallimbsofhorsesundergoaprolongedandoftenineffectualacuteinﬂammatoryphase,char-acterisedhistologicallybylownumbersofpoorlyarrangedmyoﬁbroblastsandfewpolymorphonu-clearcells.35,36Suchwoundsappeartorelymoreoncontracturethanre-epithelisationforsecond-intentionhealing.37ThisprocessisimpededbyEGT.ManyfactorsareimplicatedinEGTformation,forexamplelowoxygentension,poorbloodsupply,animbalanceinpro-inﬂammatorycellularsignallingmolecules(Transforminggrowthfactorbeta1(TGF-B1)andHypoxia-induciblefactor1-alpha(HIF-1a))2andthepersistenceofhaphazardlyarrangedﬁbrindepositions.35Despitegranulationtissuebeingaprominenthistologicalfeatureinallwoundsonday25(LBAC9/9andsaline9/9),thehaphazardarrangementofcollagenﬁbreswasidentiﬁedinonlyoneoftheninewoundstreatedwithLBACandtwooftheninewoundstreatedwithsaline(Table2).Additionally,nogradeIVmacroscopicgranulationtissuewaspresent(Table3),andallwoundswereatanacceptablestageofhealingbyday25(TableA1).ThelowincidenceofEGTislikelytheresultofstudydesign.Allwoundswerecreatedasepticallyandremaineduncontaminatedthroughoutthestudy.EGTisacommoncomplicationduringthemanagementoflowerlimbwoundsinhorsesduetothehighrateofwoundcontamination.2Thepresenceofwoundcontaminationappearstobeanimportantfactorwheninvestigatingnoveltopicaltherapies.Extensiveworkbythesameresearchgroupdocumentedthevariabilityinhealingratesbetweencontaminated38anduncontaminated39woundstreatedwithvariouspreparationsofmanukahoney.Therefore,itisrea-sonabletosuggestthattheeffectsofLBACmaydifferifappliedtocontaminatedwounds,anditremainsunclearwhetherLBACpreventstheformulationofEGT.ThisisacommonshortcominginexperimentalmodelsinvestigatingEGTformationandisdiscussedindetailbyAnantamaetal.1Thevolumesusedforsalinetreatmentwereinsuf-ﬁcienttolavagethewoundorsigniﬁcantlyalterthemicroclimatepH,meaningthattheeffectsofsalineasadecontaminantwerelikelyminimal.However,inadditiontothepre-emptivenociceptivemodu-lationbydetomidinehydrochlorideandlignocainehydrochloride,salinetreatmentmayhavereducedthepotentialdifferenceinantinociceptiveandwoundhealingeffectsbetweenLBACandsaline.Thismayreducethepertinenceofthepowercalculation.Localanaestheticsareextensivelyusedinveteri-naryhospitalstoprovidelocaltissuedesensitisationpriortoandfollowingsurgicalintervention.Despiteclaimsofreducedtensilestrengthandincreasedleukocytictrafﬁckingfollowingtheuseofsomelocalanaesthetics,datafrommurineandlagomorphmodelsindicatethatlidocainehydrochlorideand

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VETERINARYRECORD 11of13 bupivacainehydrochlorideresultinnoadverseeffects.40–42However,thetopicaluseoflido-caine‒adrenalineislesswelldeﬁned.Inrats,thesubcutaneousinﬁltrationoflidocaine‒adrenalinetransientlydelayedwoundhealingonday7andresultedinthedelayedrearrangementofcollagenﬁbres.43AsimilarphenomenonmayhaveoccurredinwoundstreatedwithLABC,withratesofhealingretardingonday11beforeacceleratingtowardsday25(Figure7).Anincreaseinwoundareabetweendays0and4isknowntooccurduringtheearlystagesofwoundhealingduetothecontractionofperilesionaltissue.Theeffectofcetrimideonﬁbroblastviabilityandwoundhealinghasnotbeeninvestigated.Medi-Solfen,asimilarproducttoTri-Solfen,containing5%(w/w)lidocainehydrochloride,0.5%(w/w)bupiva-cainehydrochloride,0.00451%(w/w)adrenalineacidtartrateand0.5%(w/w)cetrimide,iscurrentlyunder-goingphasetwoclinicaltrialsforuseonacutelytrau-maticskinwoundsinhumans.Theresultsfromtheseclinicaltrialsmayprovideadditionalinformationinthefuture.Onehorsedevelopedwhitelinediseaseonday13duringtheturn-outperiod.Whiletheonsetofwhitelinediseasecanbeinsidious,thediseasewasmildandthehorserespondedwelltosupportivemanage-ment.Anypossibleimpactonwoundhealingduetounevenweightdistributionwasconsideredunlikely.Additionally,thediseasewasnotapparentduringtheMTtestingperiodandthehorsedidnotdemon-strateanyphysiologicalorbehaviouralidentiﬁersfordiscomfort.Finally,woundhealingassessmentsonday25wereperformedbetweendays23and27dependingonthedayoftheweek.Theimpactondatainterpretationwasconsideredminimal,aseachhorseofferedoneLBAC-treatedandonesaline-treatedwound.CONCLUSIONSThetopicalapplicationofLBACimmediatelyfollow-ingwoundinganditsreapplication24hourslaterincreasedtheaveragemechanicalforcerequiredtoelicitabehaviouralresponseonday1.TheapplicationofLBACondays2and3didnotresultinclin-icallyappreciableantinociception.Additionally,theuseofLBAConcedailyfor7daysdidnotadverselyaffecttherateofwoundhealingoverthe25-daystudyperiod.However,furtherresearchisrequiredtoinvestigatetheantinociceptiveandwound-healingeffectsofLBAConnaturallyoccurringpost-traumaticwoundsinhorses.AUTHORCONTRIBUTIONSAlbertSole-Guitart,JoanneRaingerandWendyGood-wincontributedtoconceptualisation,methodology,investigation,softwareandwriting—reviewandedit-ing.WendyGoodwinadditionallycontributedtodatacuration,supervision,projectadministration,resourcesandfundingacquisition.KarladeKlerkcontributedtotheinvestigation,datacuration,for-malanalysisandwriting—originaldraftpreparation,reviewandediting.ChiaraPalmiericontributedtodatacuration,formalanalysisandwriting—reviewandediting.ShaunPrattcontributedtoformalanalysis,investigation,datacurationandwriting—originaldraftpreparation,reviewandediting.Eliz-abethEvansandJaneHumecontributedtocon-ceptualisation,methodology,projectadministrationandwriting—reviewandediting.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.ACKNOWLEDGEMENTSTheauthorswouldliketoacknowledgethefollowingindividualsfortheirextensivecontributions.Veron-icaSmithfromMedicalEthicsforprovidingproductinformationandstatisticaladvice.MitchCoyleandRebeccaCameronfromtheUniversityofQueenslandSchoolofVeterinaryScience’sEquineUnitfortheirassistancewithanimalmanagement.JoGordonfromtheUniversityofQueenslandSchoolofVeterinaryScience’sPathologyDepartmentfortissuehandlingandpreparation.BiancaAmietfromInvetusforstudymanagement.ThestudywasfundedbyInvetus(grantrecord2018002127).CONFLICTOFINTERESTSTATEMENTThestudywassponsoredbythemanufacturerofthemedication—AnimalEthics.Invetuswasthevet-erinarycontractresearchorganisationthatimple-mentedthestudyinconjunctionwiththeUniver-sityofQueensland(UniversityofQueenslandgrantrecord:Invetus2018002127).AuthorsE.E.andJ.H.wereemployeesofInvetusduringtheexperimentaldesign,conductandanalysisofresults.Thedatainter-pretationandpreparationofthemanuscriptwereassistedbyVeronicaSmith,anemployeeofMedicalEthics.DATAAVAILABILITYSTATEMENTThefulldatasetcanbemadeavailableuponrequesttothecorrespondingauthor.ETHICSSTATEMENTAllprocedureswereperformedwiththeapprovaloftheUniversityofQueensland’sProductionandCompanionAnimalEthicsCommittee(SVS/300/18/INVETUS).Informedconsentwasobtainedfromtheownerortheauthorisedagentfortheowneroftheanimalsenrolled.REFERENCES1.AnantamaNA,DuCheyneC,MartensA,RothSP,BurkJ,DeSpiegelaereW,etal.Thegranulation(t)issue:anarra-tiveandscopingreviewofbasicandclinicalresearchoftheequinedistallimbexuberantwoundhealingdisorder.VetJ.2022;280:105790.2.TextorJA,ClarkKC,WalkerNJ,AristizobalFA,KolA,LeJeuneSS,etal.Allogeneicstemcellsaltergeneexpressionandimprovehealingofdistallimbwoundsinhorses.StemCellsTranslMed.2018;7:98‒108.

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VETERINARYRECORD 13of13 bufferonwoundhealinginratskin.WoundRepairRegen.2011;19:223‒28.SUPPORTINGINFORMATIONAdditionalsupportinginformationcanbefoundonlineintheSupportingInformationsectionattheendofthisarticle. Howtocitethisarticle:PrattS,Sole-GuitartA,deKlerkK,EvansE,HumeJ,PalmieriC,etal.Antinociceptiveandwoundhealingeffectsofacommercialformulationoflidocaine,bupivacaine,adrenalineandcetrimideappliedtopicallytosuperﬁcialskinwoundsinhorses.VetRec.2024;e4395.https://doi.org/10.1002/vetr.4395

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