studies on the effect of various topical agents on second

Studiesontheeffectofvarioustopicalagentsonsecondintentionwoundhealingoftheequinedistallimb.

VictoriaCMcIver

AthesissubmittedinfulfilmentoftheMastersofVeterinaryClinicalStudiessubmittedSeptember2020.

FacultyofScienceUniversityofSydneySydney,Australia

StatementoforiginalityThisthesiscontainsmaterialpublishedinAustralianVeterinaryJournal,2020.ThisisthroughoutthethesisbutparticularlyChapters3and5.ThispaperpublishedtheresultsofPart1ofthetrial.MyinvolvementwasthatoffirstauthorandIwasinvolvedinstudydesign,implementation,obtainingandanalysingdataandauthoringofthepaper:

McIver,VC,Tsang,AS,Symonds,NE,Perkins,NR,Uquillas,E,Dart,CM,Jeffcott,LB,Dart,AJ(2020)Effectsoftopicaltreatmentofcannabidiolextractinauniquemanukafactor5manukahoneycarrieronsecondintentionwoundhealingonequinedistallimbwounds:apreliminarystudy.doi:10.1111/avj.12932Thisistocertifythattothebestofmyknowledge,thecontentofthisthesisismyownwork.Thisthesishasnotbeensubmittedforanydegreeorotherpurposes.

Icertifythattheintellectualcontentofthisthesisistheproductofmyownworkandthatalltheassistancereceivedinpreparingthisthesisandsourceshavebeenacknowledged.

VictoriaCMcIver

As supervisor for the candidature upon which this thesis is based, I can confirm that the authorship attribution statements above are correct.

Andrew Dart 16th September 2020

TableofcontentsListoffigures………………………………………………………………………………………………….4Listofappendices…………………………………………………………………………………………...5Listofabbreviations………………………………………………………………………………………..6Chapter1:Abstract…….……………………………………………………………………………………6Chapter2:Literaturereview……………………………………………………………………………8 2.1Asummaryofwoundhealing..………………………………………………………...8 2.2Complicationsofwoundhealinginthehorse………………………………….13

2.3Asummaryoftopicalwoundtherapies…………………………………………..162.4Asummaryoftheuseofmedicalmarijuana…………………………………..212.5Asummaryofthetherapeuticuseofhoney…………………………………...242.6Modelsofwoundhealinginthehorse…………………………………………….28

Chapter3:Apilotstudyintotheeffectsoftopicaltreatmentofcannabidiolonsecondintentionhealingofequinedistallimbwounds…………………………………...31

3.1Introduction………………………………………………………………………………….31 3.2Materialsandmethods…………………………………………………………………..31 3.3Results…………………………………………………………………………………………33 3.4Discussion…………………………………………………………………………………….35Chapter 4: A study into the effect of incremental increases of UMF ratings ofmanukahoneyandMelaleucahoneyonsecondintentionhealingofwoundofthedistallimbofthehorse…………………………………………………………………………………..39 4.1Introduction………………………………………………………………………………….39 4.2Materialsandmethods…………………………………………………………………..40 4.3Results………………………………………………………………………………………….41 4.4Discussion…………………………………………………………………………………….42Chapter5:Conclusionsandfuturedirections…………………………………………………46Bibliography…………………………………………………………………………………………………47Appendices……………………………………………………………………………………………….......63

LISTOFFIGURESFigure1:Plotofmeanwoundareaforeachgroupoverthe42daysofthestudy.Barsrepresent95%confidenceintervals.Figure2:Lineplotofrawwoundareameasurementsanddisplayingonelineperhorse.Thelineforhorse#4islabelled.Figure3:Plotofmeanwoundareaforeachgroupoverthe42daysofthestudy.Barsrepresent95%confidenceintervals.

LISTOFAPPENDICESAppendix1:AnimalethicsAppendix2:AnaestheticmonitoringAppendix3:HorserecordsAppendix4:Post-operativemonitoring

LISTOFABBREVIATIONSCannabidiol CBDCyclooxygenase COXInterferongamma IFN-ΥInterferonregulatoryfactor IRFInterleukin ILLipoxygenase LOXMethylglyoxyal MGOMetalloproteinase MMPMinimuminhibitoryconcentration MICMitogenactivatedkinases MAPMulti-drugresistantStaphylococcusaureus MRSANitricoxide NONon-peroxidaseactivity NPANon-steroidalanti-inflammatory NSAIDNuclearfactor NF-κβ,Plateletrichplasma PRPTissueinhibitorsofmetalloproteinase TIMPTetrahydrocannabinol THCToll-likereceptor TLRTransforminggrowthfactor-β TGF-βTumournecrosisfactorα TNF-αUniquemanukafactor UMFVascularendothelialgrowthfactor VEGF

CHAPTER1:ABSTRACTTraumaticwoundsofthedistallimbarecommoninhorses.Thesewoundsareoftenlefttohealbysecondintentionbecauseofsubstantialtissueavulsionandcontamination.Inthehorsesecondintentionhealingisoftenproblematicandwoundsaresusceptibletotheproductionofexcessivegranulationtissue,whichinhibitstheprogressionofhealing.Therehavebeenmanyproductsrecommendedtoenhancesecondintentionwoundhealinginhorses,howevernoproducthasprovedtobeatreatmentpanacea.Marijuana(Cannabissativa)containsmorethan60biologicallyactivechemicalagentsthathavebeenshowntohavepotentantibacterialandanti-inflammatoryactivityaswellastheabilitytoimproveelementsofhealinginaratskinwoundhealingmodel.Thecurrentstudyaimedtoinvestigatetheeffectsofoneresin,cannabadiol,onsecondintentionwoundhealinginhorses.Thefirstpartofourstudywastodetermineifdailytopicalapplicationofcannabidiol,incombinationwithacarrierofmanukahoney,wouldimprovewoundhealingvariablesinanequinemodelofsecondintentionwoundhealing.Severalstudieshaveshownmanukahoneyenhancessecondintentionwouldhealinginhorsesinequinewoundhealingmodels.Astheuniquemanukafactor(UMF)increasessodoestheantimicrobialactivityofthemanukahoney.MoststudieshavefocusedonUMF20honeyincontaminatedanduncontaminatedequinewoundhealingmodels.ThesecondpartofourstudyexaminedtheeffectofdifferentUMFhoneyonsecondintentionwoundhealing,andcomparedthistotheuseofMelaleucahoney.OurhypothesiswasthatincreasingtheUMFratingwouldimprovewoundhealingandthatMelaleucahoneywouldnotenhancesecondintentionwouldhealinginthehorse.

CHAPTER2:LITERATUREREVIEW2.1AsummaryofwoundhealingWoundspresentacommonchallengetoveterinariansandmedicalprofessionals.Inequinepracticethetreatmentofwoundscanquicklybecomeexpensiveduetobandagingcostsand,forperformancehorses,timelostfromtraining.Therefore,understandingtheprocessofwoundhealinganddevelopingtechniquesandtreatmentstooptimisetheprocessofwoundrepairisimperativeforequinepractitioners.Woundscanbeclassifiedasopenorclosed.Openwoundsarethosethatbreachtheskinbarrierduringtheinjuryprocess.Thesecanincludelacerations,abrasionsorsurgicallycreatedincisions.Incomparison,closedwoundsdonotbreachtheskinsurfaceandmayincludecrushinginjuries(1).Whenassessingthedegreeofbacterialcontamination,woundscanbecategorisedintoclean(surgicallycreated),clean-contaminated(surgicallycreatedwoundsenteringintotheurogenital,respiratoryorgastrointestinaltract),contaminated(eithersurgicallycreatedwoundswhereamajorbreachinaseptictechniquehasoccurred,oropen,traumaticwoundswithgrosscontamination),ordirtywounds(olderwounds,thosewithseveregrosscontamination,orthosewithnecrotictissuepresent)(1).Cleanorclean-contaminatedwoundshavelessthan1x105bacteriapergramoftissue,whilethosethataredirtyhavegreaterthan1x105bacteriapergramoftissue(2).Inhumanstimefrominjurytopresentationisusedtoclassifywoundsbecauseadelaytimetopresentationtypicallyequatestogreatercontamination(3).Inequinepractice,mostwoundsseenbyequinepractitionersareconsidereddirty.Insomecasesthisisduetothechronicityofthewounds,butmoretypicallyitisduetotheseverecontaminationpresent(1).Thisdegreeofcontaminationisdifficulttoavoidgiventheenvironmentthathorsesarehousedin.Duringwoundhealingthetissueundergoesaseriesofdifferentstagesincludinginflammation,proliferation,andfinally,remodelling(1).Thisprocessisacontinuumandeachstageoverlapsthenext.Regardlessoftheinitialclassificationofthewoundallopenwoundsfollowthisprocessduringhealing,howeverthebodyadaptstheprocessinanattempttomaximisethehealingoftheindividualwound.InflammatoryphaseTheinitialreactiontoawoundisinflammation.Theinflammatorystageinitiatestheprocessofhealingbyeradicatingcontaminantsandforeignmaterialsandprovidesthefoundationforthesubsequentstagesofhealing.(4).

Initiallyhaemostasislimitsbloodlossthroughvasoconstriction.Theinjuredendothelialcellsreleasephospholipids,whicharetransformedintoarachidonicacid,beginninganinflammatorycascadeandreleasingendothelinandthromboxaneA2(4).Thiscausesplateletaggregationandmediatesthevasculartoneofthearea(4).Thislastsonlybriefly,approximately5-10minutes,beforevasodilationbeginsinthesmallvenules(4).Diapedesisofcells,fluidsandproteinsoutofthevesselsoccursandtheseaccumulatewithintheextravascularspace(4).Aplateletplugdevelopsthroughthecoagulationprocess(4)andsofttissueswellingalsoprovidessomevascularcompression,furtherfacilitatinghaemostasis(5)(6)(7).Thisplateletplugultimatelystabilisesthewoundsiteandbecomestheprovisionalwoundmatrix,whichwilllaterprovideaphysicalscaffoldduringhealing(4).Theplateletsthatareactivatedduringthisearlystageofhealingarepotentandpromoteinflammationandreleasechemoattractants.Thisprovisionalmatrixwilleventuallydesiccate,ifleftuncovered,andultimatelybecomesascab(1).Thisscabprovidesaprotected,moistenvironmentandlimitsmicrobialpenetrationintothewound.Intheearlyinflammatoryphaseactivatedplateletsandmastcellsandinjuredoractivatedmesenchymalcellsinitiatethecomplementcascadeandpromoteleukocytesfromnearbycirculatingpoolstomigrateintothewound(4).Thisinitiatesastep-by-stepprocessofactivation,tightadhesionandtransmigrationofleukocytesintotheareaviatheendothelium(4).Chemoattractantsstimulatethereleaseofenzymesfromneutrophils,whichimprovestheirpenetrationthroughvascularmembranes.Thevasodilationalsofacilitatescellmigration.Overalltheresultisamassiveinfluxofneutrophils,beginningwithinminutesofinjuryandpeakingontheseconddayafterinjury(4).Theroleoftheneutrophilsistwofold;toclearthewoundsiteofforeignmaterialandbacteria,andtoreleasechemoattractantsthatwillfurtherdrivetheinflammatoryphase(8).Neutrophilsacttodestroybacteriaandremoveforeignmaterialbyphagocytosisandenzymaticdegradationandtheproductionofoxygenradicals(4).Proteinasesarealsoreleasedtoremovedamagedcomponentsoftheextracellularmatrix,andtheseincludeprincipallyneutrophilspecificinterstitialcollagenase,neutrophilelastaseandcathepsinG.Theneutrophilsareconfinedwithintheclotandtheiractivityceasesoncetheareaisclearedofcontamination.Thisclotwilllaterslough.Anyremainingneutrophilswillbephagocytisedbymacrophagesormodifiedwoundfibroblasts(4).Neutrophilicinfluxintothewoundisshortlived,asaretheneutrophilsthemselves(4).Afterthesecondday,monocytesbegintoexitthevasculatureandenterthewound,transformingintotissuemacrophages.Theyadheretotheextracellularmatrixandtransformintoaphenotypethatallowsthemtoproducecytokinesthatbothsupporttheirownexistenceandstimulatethewoundrepairprocess.Theysecreteproteinases,includingelastase,collagenaseandplasminogenactivator(PA)(4)whichacttoclearthewoundofremainingdebrisbeforerepairbegins(4).Notonlydotheyclearthedebris,theyalsocontributeto

recruitmentofothercellpopulations.Thesecellslateracttocreatenewbloodvessels(angiogenesis)andstimulatefibroplasiaandepithelialisation(4).Thewoundrepairprocessisorchestratedbycytokinesthatworkinunisontoguideandorganisethewoundrepairprocess(9).Whileamultitudeofcytokinesareinvolved,transforminggrowthfactor-β(TGF-β)hasthebroadestactivity(4).AwiderangeofcellsproducethreedistinctisoformsofTGF-B(TGF-B1,TGF-B2andTGF-B3),andexpressionofthetwomostrelevantisoforms(TGF-B1,TGF-B2)isparticularlyimportantduringearlythestagesofhealing(10).TGF-β1isfoundinmosttissues(4)andwhilethereisaninitialreleaseofthiscytokinefrominjuredtissues,continuedmodulationofit’sreleaseisdeterminedbyauniqueabilitytoregulateitsownsynthesisfrommonocytesandmacrophages(4).TheoutcomeofthisisaconstantsupplyofTGF-β1duringthewoundrepairprocess(4).Themajoreffectofthiscytokineistoactasachemoattractantformonocytesandmesenchymalcells,particularlyfibroblastslaterinthehealingprocess(4)(10).Whilstneutrophilsareconsideredbeneficialtowoundhealing,theyarenotstrictlyrequiredforhealingtoproceedinasepticwounds(11).Thismaybedifferentincontaminatedwounds.Incontrast,macrophagesareconsideredthemajorcellresponsiblefordebridementofwoundsand,comparedtoneutrophilswhoseroleisstrictlytoclearthewound,macrophagesalsohavearoleinstimulatingwoundrepair(12)(4).Withoutmacrophages,studieshavedemonstratedthatthereisaseverelimitationintheabilityofclearingdebrisandadelayintissuefibrosis(13).Inhorses,specifically,neutrophilicinfluxisslowerandoverallneutrophilnumbersremainhigherandforaprolongedperiodresultinginchronicinflammation(14).Incontrast,thisisnotseeninponies.Theleukocytesofponiesproducegreatervolumesofcytokines,chemoattractantsandgrowthfactors.Overallhorseshavebeenshowntohaveaprolongedandsub-optimalinflammatoryresponsewhencomparedtoponies.Experimentalstudiesexaminingwoundhealingonthemetatarsusandbuttockshavedemonstratedthatbothprimaryandsecondaryintentionwoundhealingisprolongedinthehorsewhileponieshadasignificantlystrongerandfasterinflammatoryresponse.Thisexplainswhyhorsestypicallyhavepoorerwoundhealingthantheirsmallercounterparts(1).TissueproliferationphaseThesecondphaseofwoundhealingistermedthetissueproliferationphase(1).Thisbeginswiththedevelopmentofnewbloodvesselsformingfrompre-existingones.Thisprocessistermedangiogenesis,andthesevesselssupplythenewtissuewithfactorsandoxygen(1).Thereisaconcurrentfibroblasticproliferationasthebodylaysdownnewtissuestofillthedefect(4).Thisstageoccursastheinflammatorystagedownregulates(4)andtheoutcomeisthecreationofgranulationtissue.FibroblastsmigrateintothewoundundertheguidanceofTGF-B1(4)tohelpformgranulationtissuealongwithmigratingmacrophagesandnewcapillarynetworks.Fibroblastsmigrateintotheareabytheseconddayafterwoundingandpredominatebydayfour(16),(17)withnumberspeakingsevento

fourteendaysafterinjury(16)(18).Fibroblastsmigratefromtheadjacentuninjuredtissueofthedermisandsubcutisbehindtheadvancingfrontofmacrophageswhichprovideapathclearedofcontaminants(4).Behindthefibroblastscomesnewcapillarynetworks(1)(4).Fibroblastsarestimulatedtoexpressintegrinreceptorstoassistmigrationintothetraumatisedarea(4).Integriniscriticaltocellmigratorypatterns(4).Fibroblastsalsosecreteproteasesthatacttoremovethefibrinclotandenhancecellmigration(4).Newcomponentsofextracellularmatrixdepositedduringfibroblastmigrationresultinarapidincreaseintissuevolume,althoughthisnewtissuelackssignificantstrength(4).ThisnewmatrixisreplacedbyaType1collagenrichsubstrateofglycosaminoglycans,fibronectinandhyaluron(4).Atthisstage,TGF-β1actsasapro-fibroticagenttolimittheturnoverofthenewstromabydownregulatingmetallomatrixproteinases(MMPS)andincreasingtheexpressionoftissueinhibitorsofmetalloproteinases(TIMPs)(4).Theresultingtissueactsasabarriertoinfectionandprovidesasurfaceacrosswhichnewepithelialcellsmaylatermigrate(4).Thedevelopmentofnewbloodvesselsisinitiatedbyanenvironmentthatisoxygenpoor,withhighlactatelevelsandlowpH(7).Pre-existingvesselsdevelopincreaseinwallpermeabilityandproteinasesfromactivatedendothelialcellsdegradethelocalbasementmembrane,whichallowsamigrationofendothelialcellsintotheinterstitialspace.Thisbeginswiththeextensionoftheircytoplasmicpseudopodiainitially,beforetheendothelialcellitselfcanfollow(4).Theremainingendothelialcellsthatdonotmigrateinsteadproliferate,providingasourceofnewcells(4).Thesenewsproutsofvesselsthenjointheneighbouringsprouts,fusingintoanarcade(4)andformingaprimitivecanalthaterythrocytescanpassthrough.Thesethenmature,becomingacapillary(4).Thisprocessbeginsontheseconddayafterinjury.Itreliesontheconcurrentformationofnewextracellularmatrixtoactasascaffoldforthesefragilevessels(4).Thesedelicatevesselscontinuetogrowinthepresenceofthecytokinesreleasedwithinthewound,inparticularTGF-β1.(4).Growthofthesevesselsceasesoncethetissueisadequatelyperfused(8).Atthisstage,stimuliaredownregulatedandthenewnetworkofcapillariesisreduced(4).Thisoccursthroughtheactionofmatrixmetalloproteinases(MMPs)(14)andapoptosisoftheendothelialcells(4).Clinicallythismanifestsasachangeintherichredcolourofgranulationtissuetothatofapalertissue(4).Afterthefirst10-14days,collagensynthesisslows(4).Capillariesregressandthegranulationtissuebeginstobereplacedbyalessvascularscartissue.Fibroblastsundergoeitherapoptosisortheyacquiresmoothmusclecharacteristicsandparticipateinwoundcontraction(4).Duringthisstage,TGF-β3becomesimportantbydown-regulatingtheinflammatorydrive,reducingaccumulationofextracellularmatrixandlimitingfibroplasia(19).Thisultimatelylimitsscarring(20).Thebarrierepitheliumprovidesgivesprotectionfromtheoutsideworldsore-epithelialisationofthewoundcommencesquickly.Indirtyorinfectedwoundshowever,epithelialisationcannotoccuruntilahealthyunderlyinggranulation

tissuebedhasbeencreated.Amoistenvironmenttypicallyspeedsmigrationanddissolutionofthescabisnotrequired(21)(22).Theprocessbeginswithin24-48hourswiththecentripetalmigrationofexistingcellsfromthesurroundingrimofskin(4)(23)(24).Insuperficialwounds,epithelialcellsareavailablefromwoundedgesaswellasthebasementmembraneandappendagepopulations(forexamplehairfollicles,sebaceousandsweatglands)(25),(26)acrossthewound.Thisacceleratestheprocessdramatically.Epithelialisationinitiallydoesnotinitiallyrequireproliferationofepithelialcells.Initiallyitreliesonexistingpopulationsofcellsmigrating(4).Indeed,TGF-βisthoughttoinitiallyinhibitproliferation,insteadencouragingmigrationofexistingpopulations(27)(4).Priortomigrating,basalepidermalcellsatthewoundedgesalterthemselvestoimprovemobilityandphagocyticabilities(4).Thereisanincreaseinproteinaseactivityincellsoftheadvancingfrontthatallowsthecellstodegradeanyexistingclotandtoclearremainingdebris(4).Thecellsthenmigrate,withtheaidofintegrinreceptorsandundertheinfluenceofTGF-β.Eventuallyproliferationbeginsandthecellsbegintoleapfrogoveroneanotherasthemigratingfrontadvances(4).Thesecellsformanadherentmonolayer.Thismonolayermaturesintoamorenormalmultilayeredepidermis(4).Oncethecellshavecoveredtheentirewound,expressionoflamininintheextracellularmatrixleadstocontactinhibitionandmigrationceases(4)(16).AnewbasementmembraneformsandnewcellularlayersattachtothebasementmembranethroughType4collagenfibrilsandhemidesmosomes(4).Thispartoftheprocesstakessignificanttime,andcontinuestooccurlongaftergrosshealinghasoccurred(4).Strengthslowlyincreasesinthewound(4).Woundhealingbyepithelisationalonewouldbeslowsowoundsundergocontractiontoenhancethehealingprocess.Afterinjurythereisaninitialphaseofretraction,duringwhichtimewoundareaincreases.Thislastsfor5-10daysafterwoundingandrepresentsthetimepriortopeakfibroblastinvasionfollowedbyaperiodofrapidcontraction(4).Duringthecontractionphase,thedermisandepidermisaroundafullthicknesswoundmovecentripetallyoverthewoundbed(4).Thisbeginsinthesecondweekafterwounding(4)anddecreasesthetotalareathatmustbeepithelialised.Contractiondependsheavilyonfibroblastandmyofibroblastpopulationswithinthewound(16).ThesepopulationsareabundantwithingranulationtissueandareinfluencedbyTGF-β(4).Myofibroblastshaveasmoothmuscleactinsystemalongthelongaxisofthecellthatisrelatetotheextracellularmatrixthroughintegrins,andthecellsthemselvesarealignedalonglinesoftensionwithinthewound.Eachcellisconnectedtotheneighbouringpopulationsbygapjunctionsandhemidesmosomes(4).Thisgivestheentirecellpopulationameanscontract,decreasingthesizeofthewound.Followingaperiodofrapidcontraction,itslowsdramaticallyandfinallyceasesoncethewoundedgesmeetortensioninthesurroundingtissuesbecomestoogreat(28)(17).Attheconclusionofcontraction,myofibroblastnumbersdecreasethroughapoptosisorreversiontoaquiescentfibroblast(4).Ultimatelythisprocesscanreducethesurfaceareaofthewoundbybetween40-80%(29).Inareasofloosetissue,contractioncanoccuratarateof0.75mmperday(30).Thisisreducedinareas

oftension.Fibroblastscontinuetoproducecollagentostrengthenthewound(17)(18)andthisoccursalonglinesoftension(28),furtherincreasingtissuestrength.TissueremodellingphaseRemodellingbeginsinthesecondweekofhealingbuthasapoorlydefinedend,onetotwoyearsafterinjury.Tissueisneverregeneratedtopre-injurystatus,insteadscartissueforms.Thisscartissuewillalwaysbe15-20%weakerthantheoriginaltissue(31).Remodellingseestheprovisionalextracellularmatrixconvertedintoscartissue.Itbeginsashyaluronintheprovisionalmatrixisreplacedbyproteoglycansintheextracellularmatrix.Fibroblastproliferationceasesovertime,asdoesmigration(17)(18).Thecollagencontentgraduallyincreases.Atthesametime,cellularcontentsdecreaseduetoreductionsincytokineproduction,signallingandneovascularisation(32).Collagenfibres,whichwereinitiallylaiddownrandomlyandprovidedminimaltissuestrength,begintoorientateinanorganisedmanner.Theyreforminbundlesalonglinesofstressandthisgivestrengthtothenewtissue(4).Collagen,nowinbundles,thencross-links(32)andintegratesintopre-existingbundles(4).Thereissimultaneousandequaldestructionandgenerationofcollagenduringthisphase,resultinginnonetgain.Type3collagenisremovedandreplacedbyType1collagenuntilthepre-injuryratioof1:4isreached(4).Degradationoccursviaproteolyticenzymes,mostlytheMMPcollagenase,stromelysinandmetallogelatinase,andthereisasimultaneousbalanceinhibitorslikealpha2-macroglobulinandalpha1-antiprotease,tissueinhibitorsofmetalloproteinases(TIMPs)(4).TIMPsmayalsostimulatepartsofwoundhealing(4).Theprocessofremodellingcantakeanywhereuptotwoyears(4).Tissuestrengthgraduallyincreasesfrom20%ofnormaltissueat3weeksto50%at12weeks.Finaltissuestrengthnevermatchesthatofuninjuredtissues,insteadreachingamaximumof80%oftheinitialstrength(32).2.2ComplicationsofwoundhealinginthehorseAlthoughallwoundsessentiallyundergothesamephasesofhealing,equinepractitionersmustbeawarethatwoundhealinginhorsesisproblematic,withhigherreportedratesofdehiscenceandothercomplicationssuchasexuberantgranulationtissue(33).Woundsonthelowerlimbsofhorsesarealsonotoriousforslowertimesofhealing(34)(35)(36).Experimentallycreatedwoundsoverthemetatarsusormetacarpushealnoticeablyslowerthananequivalentwoundonthebodyofthehorse.Thisisattributedtodevelopmentofexcessivegranulationtissue,or“proudflesh”,onthelowerlimbs(34)andslowerreepithelialisationandcontraction(34)(35)(36).Thisformerislinkedtothesuboptimalinflammatoryresponsethathorsesdisplayduringwoundhealing(37).Exuberantgranulationtissuemanifestsclinicallyasa

mushroomingbedofgranulationtissuerisingabovethesurroundingskinedgesthatlimitstheabilityofthebodytocontractandepithelisethewound(1),resultinginapoorlyornon-healingwound.TheTGF-βcytokinefamilyplaysacriticalpartofregulatingwoundrepair.TGF-B1isconsideredtobethepro-fibroticisoformanditsmainroleistoattractandguidefibroblastsduringtheproliferativestageofwoundhealing.Itpeaksearlyinwoundhealing.Inthedistallimbofthehorse,TGF-B1hasbeenshowntohaveanextendedperiodofexpression(19)andmaybeassociatedwiththeprofibroticandpro-inflammatorythatunderpinthedevelopmentofexcessivegranulationtissueinthehorse(31).DifferencesinthespatialandtemporalexpressionofTGF-B1andTGF-B3betweenbodyanddistallimbwoundsmayalsoinfluencehealingpatterns(31).TGF-B3isexpressedearlierandinhigherconcentrationsinwoundsofthetrunkthanthoseofthelowerlimbsandmayhelpexplainwhywoundsofthebodyhealinamoreefficientlythanthoseofthelowerlimbs(19).Bandagingoflowerlimbwoundsmayfurthercontributetoapro-inflammatorystatebytrappinginflammatorymediatorsagainstthetissuesofthewound(1).Tissuehypoxiamayalsocontributetodysregulatedhealing.Tissuehypoxiaisanormaloccurrenceduringtheearlystagesafterwounding,associatedwithdamagetothelocalvasculatureandtheimmediatevasoconstrictionthatoccursinanattempttolimithaemorrhage(36).Arelativehypoxiafollowsdespitevasodilationandsubsequentangiogenesisduetotheincreasedmetabolicdemandsofhighlyactivecells(36).Complicatinghypoxiainthehorseistheinherentlylowerbloodflowofthelowerlimb,eveninareaswithoutinjury(36).Occlusionofthemicrovasculaturehasbeenshowntooccurmorecommonlyindistallimbwoundscomparedtowoundsoftheupperbodyonthehorse(38).MicrovasculatureocclusionmayupregulateTGF-B1,encouragingaprofibroticstateandexplainingwhydistallimbwoundsarepronetothiscomplication.Hypoxiainherentlyincreasesangiogenesisandproliferationoffibroblasts–thetwomaincomponentsofgranulationtissue(38).Experimentalstudieshavealsodemonstrateddecreasedoxygensaturationandanincreaseinglucoseutilisationandlactateaccumulationinthedistallimbofhorseswithwoundsthatdevelopexuberantgranulationtissue(36).Theauthorsconcludedthatthiswasnotsimplyassociatedwithhypoperfusion,insteadtheysuggestedthatoxygendiffusionoraerobicglycolysiswasimpaired(36).Thesestudiesdemonstratedincreasedurea-adjustedlactatelevelsinexuberantgranulationtissue,whencomparedtohealthybedsofgranulationtissue.(36).Tissueoedemaorfibrosiswasproposedtobetheunderlyingcause(39).Whilerapidcellulargrowthandoxidativeburstsofwhitebloodcellscanleadtointermittentincreasesinlactateproduction(40),thelactateconcentrationsinbodywoundsconsistentlyremainedintherangeof3mmol/L,whilelactateconcentrationinlowerlimbwoundswasconsistentlyelevatedbetween4-6mmol/Lforthe38dayperiodofthestudy(36).Systemicplasmalactatelevelsremainednormal,whichconfirmedtheabnormallactateofthelowerlimbwoundswasduetolocalproductionoflactate(36).Elevatedlactatelevelsofthelowerlimbinhorseswithexuberantgranulationtissuehasbeenimplicatedasthedrivingforceforthedevelopment

ofexuberantgranulationtissue(36).Lactatehasadirectupregulatingeffectonvascularendothelialgrowthfactor(VEGF)possiblyduetothedamagingeffectsoncellsthatareresponsiblefortheirproduction(41).Italsoupregulatesgrowthfactorsandfibroblastsleadingtoangiogenesis(42)andcollagendeposition(43).Clinically,hypoxiainthelowerlimbofthehorsecanbeexacerbatedbytheroutinepracticeofbandagingthelowerlimbafterwounding.Bandagesarecommonlyappliedtolowerlimbwoundstolimitenvironmentalcontaminationandfurthertraumaandtorestrictmovementandprovidetheareawithwarmthandmoisture(44).However,bandaginghasalsobeenlinkedtothedevelopmentofexuberantgranulationtissue(44)(19),(45)(46).Experimentalstudiesusinguncontaminatedwoundmodelsofhealinginthehorseshowedthatbandagedwoundsofthelowerlimbretractmoreandaremorelikelytodevelopexuberantgranulationtissue.(44).Howeverifbandagesareremovedonceahealthybedofgranulationtissuedevelopsthetendencytodevelopexcessivegranulationtissueislimited.(1).Thediscoveryofbiofilmsinthe1970’shasdramaticallychangedtheunderstandingofwoundmanagement,particularlyinchronicwounds.Inhumanmedicineitisestimateduptohalfofchronicwoundshaveabiofilm(47).Therehasbeensubstantialresearchintobiofilmsinhumanwounds,butcurrentlythereisstillashortageofequineresearch.Abiofilmisproducedwhenbacteriabecomeencasedinalayerofextracellularmatrix,termedaglycocalyx(1).Althoughtypicallyconsideredapathogeniccondition,initialworkinthe1970’sindicatedbiofilmsmayoccurinhealthywounds,andthisworkdemonstratedtheymightactuallylimitthepenetrationofpathogenicbacteriadeeperintowounds(47).Itisimportanttonotethatawoundmaybevisiblyhealthyoruninfected,andyetstillhaveabiofilmpresent.Inapathogenicbiofilm,apolymicrobialfilmcoatswoundsorimplantsandbecomeproblematic.Biofilmslimittheaccessofantibioticstothebacterialcolonies.ExperimentalstudieshavedemonstratedthatbiofilmsofS.aureuscanbeuptoonehundredtimesmoreresistanttoantimicrobialscomparedtouncontainedbacteria.Furthermore,biofilmsincreasetheriskofanegativewoundculture,whichisclinicallyrelevanttothepractitioner(48).Theyalsotrapaconstantsourceofinflammatorymediatorsinthearea(49),leadingtoalow-gradepersistentinflammatorystate(50).Thisresultsinwoundsthatareproblematictotreat,increasingmorbidityandmortality.Inhumanmedicine,thepresenceofabiofilminwoundsisclinicallymanifestedasanimpairmentofgranulationandepithelialisation(50).Itislikelytobesimilarinahorse,howevertherearelimitedexperimentalstudiesavailable.Onestudyexaminedunbandagedwoundsoftheshoulderaswellasbandagedandunbandagedwoundsofthelowerlimbsofthehorseforbiofilms(51).Thewoundsweresurgicallycreatedandthenunderwentserialexaminationsforbiofilmformation.Theauthorsfoundthatshoulderwoundshealeduneventfullywithoutevidenceofbiofilmformation.Incomparison,alllowerlegwoundsthatwerebandagedbegantodevelopabiofilmatday7-10andbyday10-14thesebiofilmswerewellestablished.Thesewoundsweremore

pronetothedevelopmentofexuberantgranulationtissue.Incontrastonly57%ofunbandagedlowerlimbwoundsdevelopedabiofilmandnounbandagedlowerlimbwoundsdevelopedexuberantgranulationtissue.Thebiofilmsinunbandagedlowerlimbwoundswerelessdeveloped.Mostsuperficialbiofilmsthroughoutthestudyculturedcocci,withonebiofilmfromdeeperwithinthewoundculturingrods(51)buttheauthorsdidnotfurthertypetheisolates.AsimilarstudyfoundthatsuperficialbiofilmstypicallyconsistedofS.aureus,whilstthoseofdeeperareastypicallygrewP.aeruginosa(52).Itwouldbereasonabletospeculateisolateswereconsistentinthetwostudies.Whybiofilmsdevelopedinthelowerlimbsandnotinbodywoundswasnotexplainedbythisstudy,howevermanyofthefactorsthatareknowntocontributetobiofilmformationalsofavourthatofexuberantgranulationtissue.Persistentinflammation,hypoxiaandhypoperfusionarefoundinbothpathologicalstates(51).Itispossiblethattheinherentsuboptimalinflammatoryresponseofthelowerlimbofthehorsefailstoproperlyclearthewoundofbacteria,allowingcolonisationandthedevelopmentofabiofilm.Itisalsopossiblethatthelowerlimbissimplyexposedtomorecontaminationatthetimeofwounding(51).Bandagingoflowerlimbsmaylenditselftobiofilmformationbecausethiscreatesamoist,warmenvironment(51).Regardless,onceabiofilmdevelopsitimpairshealing.Thebiofilmitselfdampenstheabilityofleukocytestodestroybacteria(53)andcontributestoahypoxicstatewithahighdemandforoxygen(54).Furtherstudiesareneededinthisarea.2.3AsummaryofwoundtherapiesInclinicalpracticewoundsarecommonlytreatedwithsystemic,regionalortopicaldrugs.Theprinciplesofemergencywoundmanagementaretooptimisethewoundbedtoenhancehealingwhilstminimisingcomplications,namelyinfection.Thereareexhaustivelistsoftherapiesavailableduringthewoundhealingprocess,anddiscussionofmanyofthemisbeyondthescopeofthisthesis.Drugsadministeredsystemicallyaretypicallygivenviaintravenous,intramuscularororalroutestargetingthebodyasawhole.(1).Regionallyadministeredagentsmaybegivenasaregionallimbperfusionorasanintraosseousinjectionandtargettheaffectedanatomicalregion(1).Topicalagentsareextremelyfocusedintheirareaofactionandusuallyhaveamuchmorelimitedpenetration(55).Thismeanstheyhavelimitedsystemicsideeffects.Thereareexceptions,butthesewillnotbediscussedfurther.Becausetopicalmedicationstargettheiractionstotheareatheyareappliedto,theymaybemorecosteffective,particularinlargeanimalpractice.Further,theyareusuallypracticaltoapplybyboththepractitionerandowner.Topicalagentscanhaveawiderangeofeffects.Someagentsmayhaveextremelylimitedroles,forexamplesomeagentsmayactasasimplebarrierfromtheoutsideworld,limitingpenetrationofmicrobesandpreventingtraumatothewoundbedduringdressingchanges(56).Othertopicalagentsmayhaveamoregeneralisedspectrumofactioncomparedtopharmaceuticaldrugs.Manukahoney,forexample,hasbothantimicrobialandanti-

inflammatoryeffectswhenappliedtowounds(57)(58).Selecttopicalagentscanalsobeusedtodebridethewoundorpromotecertainstepsinthehealingprocess.Topicalagentscanthereforehavemultipleeffectsonwounds,andoftenhaveeffectsthatextendbeyondthesimplepharmaceuticaleffectsthatdrugssuchasNSAIDorantibioticsoffer.(59).Itisnotjustpuretopicalmedicinalagentsthathavebeenshowntoalterwoundhealing.Inthedevelopmentofequinecontaminatedwoundmodelsthetopicalapplicationoftopicalfreshfaecestoasurgicallycreateddistallimbwoundfor24hourswasfoundtoleadtogreaterretractionofthewoundandfasterhealingwhencomparedtouncontaminatedwounds.Thisislikelytobeassociatedwithupregulationoftheinitialinflammatoryresponsethatisoftensuboptimalinequinedistallimbwounds(60).Thisdemonstratesthevariedwaysinwhichtopicalagentscanhaveaneffectonopenwounds.Topicalagentsincludecreams,ointmentsanddustingpowders.Theseformulationscarrythechosenactiveingredientthroughtheskinbarrierorwoundsoboththeformulationandactiveingredientmustbeappropriateforpenetrationtooccurtothedesireddepth(61).Someagentsmaylimittheireffecttothestratumcorneum,whereasothersmaypenetrateintothedeeperlocaltissues.Regardless,theaimistohaveatargetedeffectontheareaofconcern(55).Despitethepositives,unfortunatelymanyoftheavailabletopicalagentssimplyfailtoperformtoexpectations,or,evenworse,inhibitwoundhealing.Muchworkhasbeendonetofindtheultimatetopicalwoundtherapeuticagentbuttodatethereisnotreatmentpanacea.Anti-inflammatoryandanalgesicagentsOneofthemainstaytreatmentsofequinewoundmanagementisprovisionofanti-inflammatorymedications.This,ofcourse,reducesinflammationbutalsopatientdiscomfort.Inequinepractice,non-steroidalanti-inflammatoriesaretypicallythedrugofchoiceforwoundtherapiesandtheyaretypicallyadministeredsystemically(1)(59).Otheragentssuchasopioidsareavailableforseverecases,butroutineusageisuncommon(1).NSAIDareusedforreductionofinflammationandpainandrepresentrelativelylowcostandeffectivemedicationsthatownerscaneasilyadministerthemselves.Whenusedappropriately,thesemedicationsaregenerallyconsideredrelativelysafe(1).Examplesincludephenylbutazone(1).However,therearesituationswheretheseNSAIDshouldbeavoided,suchasinanimalswithcompromisedrenalhealth(1)orcompetitionanimalsundergoingdrugscreening.Inthesesituations,othermodesofapplicationoralternativetherapiesmaybeconsidered.Whenfacedwithawound,practitionerstypicallyuselocalanaestheticsfordesensitisationoftheareaforthepurposesofdebridementorwoundrepair(1).Howevertopicallocalanaestheticshavealsobeenusedinhumanmedicineforthepurposesofpost-operativeanalgesia,withtheaimofreducingopioidusage(62).However,thelong-termeffectoflocalanaestheticsonwoundhealingisunclear(59).Whilesomestudiesshownonegativeeffectsofjudicioususeoflocalanaestheticsforulcerdebridementinhumans(63),animalmodelshaveshownhigherconcentrationsoflignocaineinhibits

epithelialisation(64).Giventhepotentialdownsides,localanaestheticsmaybebestlimitedtoacutepainmanagement.Unfortunatelytopicalformulationsofnon-steroidalanti-inflammatorieshavelimitedavailabilityandtheirformulations,suchasdiclofenac,arenotrecommendedbymanufacturersforuseonopenwounds(65).Topicalcorticosteroidsmaybeutilisedjudiciouslythetreatmentofexuberantgranulationtissue.Inthiscase,topicalcorticosteroidsmaybeappliedandresultindownregulationofthepro-fibroticTGF-β1,ultimatelylimitinglocalinflammationandfibroplasia(4).Whilebothfactorsareimportantinthedevelopmentofexuberantgranulationtissue(4),evidencesuggestscorticosteroidsmayinhibitangiogenesisandepithelialisation(66).Thereforejudicioususeandappropriatetimingisrequired.GiventhelimitedNSAIDsorviablelong-termalternativesavailablefortopicalusemorenovelagentssuchasCBDhavebeentrialled.Inpart,theaimhasbeentoreduceopioiddependenceinhumanmedicine.Thesehaveshownanti-inflammatoryeffectswithpromisingresults(67).Topicalagentsalsoworktoindirectlyreducepatientdiscomfortassociatedwithwoundmanagement.Muchofthediscomfortassociatedwithwoundscomesfrombandagechanges.Minimisingthiscanbeachievedbycreatingamoistwoundenvironment.Thisisalsobeneficialtowoundhealing(1)butimportantlycreatesanon-stickenvironmentandreducestraumatotissuesduringdressingchanges(68).Agentsthatcreateosmoticgradientsinwounds,suchasmanukahoneyachievethisgoal(69)(70).Thereforetheyhavesignificantvalueforthispurpose.AntimicrobialagentsOneoftheprinciplesofwoundmanagementistopreventorreducebacterialloadorresolveinfection.Theefficacyofanytreatmentisreflectedbyareductionincolonyformingunits(CFUs)(59).Mechanicalwounddebridementisencouragedasafirstlinestep,butalonethisisnotalwayseffective(1).Thereforeothermeansshouldbeused,andthismayincludetheuseofsystemic,regionalortopicalantibioticuse.Thedecisiontousesystemicantibioticsismultifactorial.Inhumanmedicine,thetimefromwoundingtopresentationislinkedtoinfectionrates.Intheequinefield,timingisrarelycriticalastheenvironmentandmanagementofhorsestypicallyprecludestheaccidentalcreationofacleanorclean-contaminatedwound(1).Instead,themechanismofwoundingandnatureofthelesionshouldbeconsidered.Forexample,aheavilytraumatisedwoundcreatedwithajagged,soiledobjectorthroughananimalbiteismoreconcerningthanasimplelacerationorsuperficialwoundwithminimalsurroundingtrauma(71).Incaseswithsignificantclinicalconcernexistsorthosewithacurrentinfection,antibioticsareoftenused(1).Thelocationofthewoundalsobecomesimportant.Systemicantibioticsarecommonlyutilisedinwoundsinvolving(orsuspectedtoinvolve)anormallysterilearea,suchassynovialstructuresorbodycavities,ascomplicationsintheseareasoutweigh

anydownsidesofsystemicantibioticuse(71).Conversely,anatomicaldifferencesinbloodflowbecomeimportant,asareasthataretraditionallylesswellperfusedmaynotreceiveadequatedosesofsystemicmedications,renderingthemlesseffective(72).Thesystemichealthoftheanimalshouldalsobeconsidered,andanimalswithcompromisedimmunestatusesorseriousunderlyingmedicalissuesmayalsobecandidatesforsystemicantibioticsastheirabilitytohealnormallymaybecompromised(71).Inessence,wherethereisseriousclinicalconcernsystemicallyantibioticsaremorecommonlychosen.Wherepossiblehowever,systemicuseofantibioticsshouldbelimitedinlinewithantibioticstewardshipguidelines.Ifsystemicdeliveryofantibioticdrugsisdeclined,regionaltherapiesmaybeelected.Theobjectofregionallimbperfusionsistodeliverhighlevelsofantibioticstoafocusedregionofconcerntoreducebacterialpopulations.Thisminimisessystemicexposureofthepatienttothedrug(73).Intravenousregionalperfusionsworkontheprinciplethatatourniquetplacedabove,andpossiblybelow,theaffectedareawillisolateitfromsystemiccirculation.Injectionofthechosendrugoccurswithintheisolatedsectionofthelimb,andthistheoreticallyprovideshighlevelsofthedrugtotheaffectedareawhilstlimitingsystemicexposure(73).However,regionallimbperfusionshavedownsides:theproceduretypicallyrequiressedationoranaestheticofthepatientandmayberelativelyexpensivetoperform,especiallywhensystemicdosagesorfrequentperfusionsareutilised(73).Furthermore,arecentreviewofthetechniquedemonstratedaninabilitytoconsistentlyachieveappropriatelevelswithintheareacutofffromthesystemiccirculation,andconverselyaninabilitytoguaranteethepreventionofsystemicexposure(73).Giventhecostandneedforaveterinariantoperformthistechnique,thismethodistypicallyreservedforthetreatmentofsynovialsepsisorforcomplicatedwoundsofthedistallimbs.Ithasimmensevalueinthesesituations.Therearelimitedintra-operativeprophylacticusesforregionallimbperfusionsofantibiotics,whichwillnotbediscussedfurther.(73).Thethirdoptionavailabletoequinepractitionersistheuseoftopicalagents.Inmanycases,systemicorregionalmedicationsaresimplyoverkill.Thereareamyriadoftopicalantimicrobialtreatmentsandlistsoftheseproductsareexhaustive(4).Evidencerelatingtotheuseoftopicalantibioticsisvariedandattimescontradictory.Onestudyfoundthatinfectedlegulcersinhumanmedicinereceivednobenefitfromtopicalantibiotics(74),whereasotherstudieshaveconsideredtheiruseiscritical(75).ArecentCochranereviewinhumanmedicinesuggeststhatthetopicaluseofantibioticsinincisionshealingviaprimaryintentionprobablyreducestheriskofsurgicalsiteinfectionafterwoundrepair,buttheevidenceismoderate(76).Thedownsidesoftopicalantibioticsincludedelayedhypersensitivityreactions(77)but,perhapsmorecritically,thedevelopmentofantibioticresistance(78).Studieshavedemonstratedthatantibioticsthathavebothsystemicandtopicalusesareparticularlypronetothis,andoneexampleofthisinequinepracticeisgentamicin(79).Withantimicrobialstewardshipbecomingincreasinglycriticalandtheevidencefortheirefficacyastopicalagentsbeinglow,topicalantibioticsshouldbereservedforselectedcases.Instead,otheragentswith

antimicrobialeffectscanbeused.Thenaturalinclinationistoturntoantisepticssuchasiodineorchlorhexidine.However,availableevidencesuggeststhatinordertoreducebacterialloads,cytotoxicconcentrationsofantisepticagentmustbeused,damaginghealthytissuesduringthisprocess(80).Hydrogenperoxideisanothercommonlycitedagents.Thishasdirectandindirecteffectsonmicrobes.ItdecreaseslocalpH,creatinganenvironmentthatisnotconducivetomicrobialsurvival,butitalsoworksdirectlybyreleasingenzymesthatdirectlydamagethemicrobe(81)(58)(82).However,hydrogenperoxidealsohascytotoxiceffectswithinwoundsbedsandretardswoundhealing(83).Itisthereforenolongerroutinelyrecommendedforwoundmanagementasaresult.Newrecommendationsthereforeadviseagainsttheuseoftheseagentsonwounds(1).Asseen,manycommonagentshavethepotentialtocausesignificantharmtofragiletissues.Thishasledtoinvestigationintoamultitudeofotheragents,suchasmanukahoney(84)ornovelagentssuchascannabidiol(85).Thesetherapeuticsaregenerallylesslikelytodevelopproblemswithresistance,givingthemaddedadvantages(86)(87).Furthermore,topicalagentsoftenhavemultipleothersimultaneousbenefitssuchascreationofamoistwoundbedoranti-inflammatoryeffects(57)(58)(88).OthergoalsThereareamultitudeofagentsavailabletothepractitionerthathavevalueoutsideofthetraditionalanti-inflammatory/analgesicorantimicrobialeffects.Theseactionsmayincluderolestargetingspecificphasesofwoundhealing,ormayhaveplacesinwounddebridement,forexample.Agentsrangefrombiologicalagentssuchasequineamnionorplateletrichplasma,ornaturalagentsorextractssuchascannabidiol,honeytosyntheticagents(89).Topicalagentscanhavefunctionsinwounddebridement.Althoughmechanicaldebridementincommonplaceinequinepractice,itcannotbeentirelyreliedupon.Enzymaticdebridementisavalidadjunctivetherapy.Enzymaticagentsincludestreptokinase,collagenase,ortrypsin.Tobeusedproperly,sharpdebridementmustbeperformedfirstastheenzymaticagentsareincapableofremovaloflargevolumesofnecrotictissue(1).Intheequinesetting,availablyandpracticalityoftheseagentslimittheiruseandasaresulttheywillnotbediscussedfurther.Otheragentssuchasgrowthfactorshavebeenusedtopicallyduringwoundhealingwithspecificgoalsinmind.Listsincludegrowthfactorssuchasplateletrichplasma,TGF-β(89)orVEGF(90).Thesemaytargetspecificphaseswithintheprocessofwoundhealing,andacttoenhancethem.Forexample,inhumandiabeticpatientsVEGFisreduced,andthisreducesangiogenesis.Clinically,thisresultsinimpairedwoundhealing.Giventhis,topicalVEGFhasbeentrialledtocorrectthisdeficiency.Andhasshownpromisingresults(90).Althoughthisisoneofmanyagentsavailable,itdemonstratesthetargetedactionofsomeagents.

ConclusionOverall,theaimofanytopicalagentistopromotewoundhealingwithnoorlimitedsideeffects,eitherlocallyorsystemically,andtolimitcomplications.Howeverthereisnotreatmentpanaceaandtherearehundredsoftopicalagentsthathavebeenutilisedinwoundhealing.Thefactthatsomanyoptionsexistsuggestsnoneareperfectineverysituation,anddemonstratestheneedforfurtherresearch.Acompletereviewoftheseagentsisbeyondthescopeofthisthesis,andassuchonlythetwomainagentsofinterest–honeyandcannabidiol-willbediscussedindetail.2.4AsummaryoftheuseofmedicalmarijuanaMarijuana(Cannabissativa),aleafyplantcommonlyutilisedasanillicitdrug,haslongbeenknowntohavetherapeuticbenefits(91).Recentlythepopularpresshasfocusedattentiononitsnumerouspotentialmedicalbenefits.Cannabiscontainsagroupofcompoundsincludingthecannabinoids,theterpenesandthephenoliccompounds(92)(93).Ofparticularinterestarecannabinoids.Thisgroupincludestetrahydrocannabinol(THC)andcannabidiol(CBD).However,over90cannabinoidsaredocumented(94).Thesecompounds,despitesimilarbiochemicalstructures,havedifferentphysiologicaleffects(94).THCmakesupthelargestproportionofcannabinoidcomponentoftheplantandhassocietalassociationsasanillicitdrug.Thisisduetoitspsychoactiveproperties,butitdoesalsohavevalidmedicinalproperties(94).CBDisthesecondlargestcomponentofCannabissativaandthemajorcomponentofhemp(94).Itisnotpsychoactive,anddoesnot,onitsown,affectmotorfunctionormemory(94).In2017,theWorldHealthOrganisationannouncedthatCBDhasnopotentialforabuseordependency(95).InterestinthetherapeuticbenefitsofCBD,whileavoidingthesideeffectsofTHC,hasincreased.Today,mostmedicalmarijuanaapprovalsareforchronicpainconditionsorformusclespasmsufferers(93).ThereisincreasinguseinneurodegenerativeconditionssuchasAlzheimer’s,multiplesclerosis,arthritisorforuseasanadjunctiveanti-nauseatreatmentduringchemotherapyadministration(93)(94).Interestingly,cannabidiolshavealsobeenshowntoexertanti-bacterialeffectsandthereforemayhavearoleininfectioncontrolasantibioticresistancecontinuestoincrease(85).Cannabinoids:MechanismsofactionTheendocannabinoidsystemisacriticalneuromodulatorypathwayfoundinmammals.Itisresponsibleforavarietyofeffectswithinthebody.Thissystemreliesonendogenouscannabinoids.Thisisasystemofenzymesproducedwithinthebodythatareresponsibleforproductionanddegradationofthesecannabinoidsandavarietyofreceptors,primarilyCB1andCB2receptors,onwhichtheyexerttheireffects.Exogenouscannabinoids,suchasthosefoundwithinCannabissativa,exerttheireffectbyinteractionswiththeseCB1andCB2receptors(96).

CB1receptorsarefoundpredominantlyinneuronaltissues,includingthebasalganglia,cerebellum,neocortex,hypothalamusandlimbiccortex(91)andalsoincentralandperipheralneurons.CB1receptorsarealsodistributedinthedorsalhornofthespinalcordandwithinimmunecellstoalesserdegree.CB2receptorsareprimarilyfoundintheperipheryofthebodyandinimmunecellsandtissues.Theyexertimmunosuppressiveandinflammatoryactivity.Toalesserextenttheyarealsofoundinthebrain(97).THCisapartialagonistoftheCB1andCB2receptorsalthoughitpreferentiallybindsCB1receptors(98).CBD,incomparison,exhibitsaloweraffinityforthesereceptorsthanTHC.CBDactsasanon-competitiveCB1andCB2receptorantagonist(99).ThisallowsittoactasanentouragemoleculetoTHC.ThisimprovesthesafetyofTHCbycounteractingsomeoftheTHCsideeffects(100)(101)(102)(103)(94).ThisisparticularlyapparentwhentheratioofCBD:THCis8:1orgreater(102)(102).InadditiontothebenefitsCBDhasinthepresenceofTHC,ithasitsownindividualmerits.Firstly,CBDhasanti-inflammatoryeffects.CBDhasaninverseagonisteffectontheCB2receptor(99),andthisresultsinananti-inflammatoryeffectbyinhibitingimmunecellmigration(104).Secondarily,itdirectlyinhibitslipoxygenase(LOX)andcyclooxygenase(COX)(105).LOXandCOXarewellknownmeditatorsoftheinflammatorycascade.CBD’santi-inflammatoryeffectsareclinicallyaremorepotentthanaspirin(106)(107,22).CBDhasalsobeenfoundtoactonglycinereceptors(108).Glycineactsasaninhibitorypostsynapticneurotransmitterofthedorsalhornofthespinalcord,andassuchCBDmayhaveaneffectinchronicpainthathasresultedfrominflammatorystatesorneuropathies.Furthermore,CBDactsasamureceptorligandandhasapositiveallostericmodulatoreffectonmuanddeltaopioidreceptors,suggestingsomeabilitytoenhanceopioideffects(98).CBDhaswidespreadandvariedpositiveeffects:anticonvulsant,antipsychotic,anxiolytic,anti-nausea,neuroprotectiveandanticancereffects(109)(110)(111).CBDandwoundhealingThereisaplethoraoftopicalagentsavailableforthetreatmentofwounds.Thesearealladvertisedasimprovingwoundhealing,eitherdirectlyorthroughindirecteffectssuchasanalgesiceffects,orbytreatinglocalinfections.GiventhehighsafetyprofileofCBD,ithasrecentlybeeninvestigatedandshowspotentialasanexcitingalternativetoothercommerciallyavailabletopicalwoundmedications.SeveralstudieshaveattemptedtodemonstratetheeffectCBDhasonwoundhealing.Thisincludesastudythatexaminedtheeffectofsystemic(specificallyintra-peritoneal)CBDonamiceoralmucosalwoundingmodel(112).Thisgroupdemonstratedasignificantreductioningrossandhistologicalinflammatoryscoresduringtheearlyphasesoftreatment.TheyconcludedthatthetopicalCBDhasapositiveeffectonearlywoundhealing,butthiseffectwaslimitedtotheearlystagesofwoundhealing.Theysuggestedfurtherresearch

wasrequiredtoproperlydeterminetheeffectCBDhad,butconcludedthetreatmentwasbothsafeandpromising(112).Theanti-inflammatoryeffectsofCBDonwoundsmayhaveapplicationtoequinepracticebecauseequinedistallimbwoundsarepronetochronicinflammationassociatedwithdysregulatedhealinginthehorse.CBDhasbeenshowntodecreaseinflammationandfibrogenesisaswellaspromotingskinre-epithelialisationinamousemodel(113).InPartCBDdecreasesinflammationdecreasingneutrophilcontent,butmoreimportantlybyhalvingmacrophagecontent.Bydecreasingmacrophagenumbers,thereisadecreaseinpro-inflammatorymediatorsreleasedsuchasinterleukin1(IL-1),interleukin6(IL-6)andtumournecrosisfactoralpha(TNF-α).Overall,inflammationdecreased.Pro-angiogenesisfactorVEGF-αisalsodecreasedwhichreducesgranulationtissuewithinthewoundwithoutalteringneovasculardensity(113).Interferongamma(IFN-Υ)activitywasincreased,andthisfactorispartiallyresponsibleforactivationofmacrophages,promotingphagocytosisandcytokineproduction.Thismayexplainwhy,despiteanoveralldecreaseinwhitecellsnumber,effectivemacrophageactivitywasabletocontinue(113).Keratinocytemigrationwasalsoimproved.(104)Otherstudieshavedemonstratedreductioninfibrosisinmicewoundingstudies(114).TheseresultsfurthersupportapotentialforCBDuseinequinewoundtherapieswherethegoalistoreduceinflammationandfibrosis.CBDalsohasapotentialantimicrobialeffectthatwouldbeusefulasawoundtherapy.RecentstudieshaveexaminedtheeffectofTHCandCBDonmulti-drugresistantstrainsofStaphylococcusaureus,orMRSA(85).Thisgroupfoundexceptionalactivityagainstsomestrainsofbacterialisolates,whichwerecomparabletoorexceedingthatofastandardprescribedantibiotic.(85).Recently,mediaoutletshavereportedsuccessbyanAustralianbasedcompanyinthetreatmentof“superbugs”andbiofilmswiththeuseofCBDimpregnatedproducts.Theseproductswereusedtopically.Accesstoresultsiscurrentlyunavailable.Itisexpectedthatthisareaofresearchwilldramaticallyincreaseinthenearfuturegiventhedevelopmentofantibioticresistance.Inhumanmedicine,painassociatedwithsignificantskinwoundshastraditionallybeenmanagedwiththeuseoftopicalorsystemicopioids.Intheveterinaryindustry,systemicopioiduseiscommon.However,therearesignificantconcernswiththeprolongeduseofopioids.Thereisawell-establishedriskofdependencyorundesirablesideeffectsinthesecases.ThishasgivenrisetotrialswithtopicalCannabidiolasanalternativemeansofmanagingpainassociatedwithskinwounds,suchasthosecausedbyPyodermagangrenosum.Althoughasmallstudy,onetrialdemonstratedclinicallysignificantreductionsinpainscoresintwooutofthreepatientswithpositiveopioidsparingeffects(67).Aclinicallysignificantreductionisgenerallyconsideredtobea30%orgreaterreductioninpainscore,asperinternationalguidelines(115).Marijuanaextractsmayhavevalueinwoundhealing,butclinicalbenefitsarelargelyunknown.CBDinveterinarypractice

Todate,literatureregardingtheuseofCBDinanimalsituationshasfocussedonexperimentalanimalmodels.ThereisextremelylimiteddataonCBDuseinveterinaryclinicalcases,withtheexceptionofthoseexaminingaccidentaltoxicity(116)(117)(118).However,thereissomereporteduseofCBDinthemanagementofglaucoma(119)(120)aswellasinthefelinedermatologyspecialtyinthemanagementofeosinophilicgranulomas(121).PublicationontheuseofCBDinthepalliativemanagementofdogsundergoingoncologicaltreatmentisalsoavailable(122),whichisnotsurprisinggiventhewell-describeduseofCBDinhumanoncologicalpatients.NopublicationsarecurrentlyavailablefortheuseofmedicalMarijuanainhorses.2.5AsummaryofthetherapeuticuseofhoneyThetherapeuticuseofhoneyasawounddressingdatesbacktothetimeoftheancientEgyptians.Ithasbeendocumentedinastaggering5000of9000recordedtherapies,suggestingaheavyrelianceontheproductthroughouthistory(123)(124).TherearealsoreportsofhoneybeingutilisedformedicinalpurposesinChinese(123),Greek(125)(126)andIslamiccultures(124)(123).Theuseofhoneythenwanedwiththeproductionofantiseptics.Theincreaseinresistancetoantimicrobialdrugshasledtoarenewedinterestinhoneyandrigorousscientificresearchhassupporteditsplaceinmodernmedicine.Recentresearchhaslargelyfocusedontheantimicrobialpropertiesofhoneyhoweverthereisanemergingbodyofevidencethatsuggestshoneymaydirectlymodulatethehealingprocess(127).Thisnewresearchalsoindicatesthatnotallhoneyvarietalsbehaveequallyandtheprofilesofthehoney’sbioactivecomponentschangethisbehaviour(128).Thereareamultitudeofvarietalsavailablefromgenerictablehoney,tothemorespecifichoneysproducedfrommanukaorMelaleucaplantspecies.CommonpropertiesofallhoneysAllhoneysarecomposedtoamixtureofsugars,aminoacidsandothersubstances.Theytypicallyhavealowwatercontent(129).ThepHofmosthoneysrangesfrom3.2to4.5,butcanextendoutto3.2to6.1(129)(58)Thisacidityisduetothegluconicacidpresentandwillcontributetotheantimicrobialeffectsofhoney(130).AlowpHenvironmentcandirectlychangetheshapeofbacterialcells(58).Thishasbeenshown,underscanningmicroscopy,tocausecelldisruptionandthelysisofPseudomonasaeruginosa(131).Furthertothis,bacterialproteaseactivityisreducedinlowpHenvironments(132).Proteasesplayrolesinthedestructionofcytokines,growthfactorsandextracellularmatrixandtheiractivityincreasestissuetraumaandretardshealing.Thisprovidesantimicrobialpropertiesbycreatinganosmoticgradientandsubsequentbacterialdehydrationandosmoticstress(127).Anosmoticgradientisalsocreatedinthewoundbed,asthereisafluidshiftfromsurroundingtissuesandvasculature.Thiscreatesamoistwoundenvironmentandsuppliesnutrientstothewound,promotingautolyticdebridementandenhancinghealing(69)(70).Currentrecommendationspromotemoistwoundenvironmentsforthesereasons(1),butalsobecausetheyminimisetraumatodelicatenewtissuesduringbandagechangesby

creatinganon-sticksurface(68).Clinicallythismayalsoreducepainanddiscomfortexperiencedbythepatientduringdressingchanges.Mosthoneysalsocontainglucoseoxidaseinlowconcentrations(129).Glucoseoxidaseactstoconverttheglucosewithinthehoneyintogluconicacidthatcausesasubsequentreleaseofhydrogenperoxide.Hydrogenperoxideisawell-knownantimicrobialsubstance(58).IthasadirectactiononmicrobesbutalsoanindirectactionthroughadecreaseinpHinhibitsmicrobesandassociateddamagingenzymes(81)(58).Thiscreatesanenvironmentthatisnotconducivetobacterialsurvival(82).Baselevelsofhydrogenperoxideactivityvarytremendouslybetweenstrainsofhoneybasedonenvironmental,plantandinsectfactors(133).Levelsaretypicallybelowdetectablelimitsinundilutedhoney(134)(135).However,whenhoneyisdilutedtheactivityofglucoseoxidaseincreasesandthisresultsinincreasinglevelsofhydrogenperoxide.Dilutionisunavoidablethroughtheadditionofawoundexudatefluid,andatdilutionsof30-50%,antimicrobialactivityisoptimised(135).Unfortunately,processingandstorage,pollenandsomebacteriamayinactivatehydrogenperoxideandthereforetheantimicrobialactionofhoneyduetohydrogenperoxideis,atbest,variable(136)(133)(137)(138).Generichoneysalsoofferanti-inflammatoryproperties.Thisoccursthroughareductionintheactivityofimportantcytokinesandfactorswithinawound.Thereisareductioninsomeofthemajorpro-inflammatorycytokines,includingTNF-aandinterleukin8(IL-8)(86)aswellasaninhibitionofMMPproduction(139)andreductioninCOXactivitythroughflavonoids,includingchrysin(140).Furthermoremacrophageproductionofnitricoxide(NO),apotentinflammatoryagent,isreduced(82)(141).Honeyhasbeenshowntobetterregulatetheinflammatoryresponsewithinthewoundaswell,byimprovingtheinflammatoryresponseofleukocytesthatproducethecytokinesthatregulateangiogenesisandfibroblastproliferation(142)(143).Inhorseswithaweakandsuboptimalinflammatoryresponse,thismayproveclinicallyrelevant.Honeyhasalsorecentlybeenusedforpreventionofbiofilmsinwounds.Topicalapplicationofgenerichoneyhasbeenshowntoeffectivelypenetrateintothebiofilm,largelyduetothesugarcomponent,whichlimitsthe“swarming”abilityofbacterialcolonies(144).Thisinturndisruptstheabilityofthecolonytoformabiofilm(145).Biofilmslimitantimicrobialpenetrationandreducethesuccessoftreatinginfections,aswellastrappinginflammatorymediatorsintheregion(49).Theabilitytotreatbiofilmsisbecomingevenmoreimportantwiththedevelopmentofantimicrobialresistance.Thereisnodocumentedantimicrobialresistancetohoney.Thisislikelyduetothemultiplepathwaysinwhichhoneyhaseffectsonbacteria(146).Ingeneralmicrobesthataresporeformingoryeastsaretheoreticallymorelikelytoberesistanttohoneybutstudieshavedemonstratedefficacyofmosthoneysagainstbacteriaisolatedfromwoundsinhorsesinvitro(146).Honeyhasbeenshowntobeeffectiveatstoppingmicrobialgrowthinvivoatconcentrationsof16%orless,evendownto4%insomecases(146).Otherpublisheddatahasdemonstratedinvitrominimuminhibitoryconcentrations(MIC)ofhoney

againstPseudomonassppat15.7%andStaphaureusat2.8%(87).Thisislowerthantheexpectedhoneyconcentrationsinawoundinvivo,eveninhighlyexudativewounds(146).Furtherstudieshaveconfirmedthatselectingforhoneyresistantmicrobialpopulationsistheoreticallypossible,butunlikely(86)(87).Thisgroupconcludedthat,inpractice,highconcentrationsofhoney(over80%)areutilised,andthereforetheyconsideredclinicalfailureofhoneyandtheemergenceofresistanceunlikely(87).Dailydressingchangesmaybeonewaytoovercomepotentialproblemsinhighlyexudativewounds.ManukahoneyTheLeptospermumspeciesofplants,morecommonlyknownasthemanukafamily,isfoundprimarilyinAustraliaandNewZealand.Mosthoneyvarietalsstudiedhavebeenproducedfromthemanukabush(L.scoparium)ofNewZealand.Honeyfromtheseplantshasadditionalbeneficialproperties,inparticulargreaterantimicrobialactivity,comparedwithgenericcounterparts(147,148).Manukahoneyalsohastheabilitytomodulatewoundhealingdirectly(57).Thesepropertieshaveledtoacommercialisationoftheproductfortherapeuticuse.Manukahoney,incontrasttogenerichoney,displaysminimalhydrogenperoxideactivityandyethasexceptionalantimicrobialproperties(58)(88).Further,hydrogenperoxidecontentdoesnotincreasewithdilution(149).Forsometimethereasonbehindthisexceptionalantimicrobialactivitywasunknownsowastermedthenon-peroxidaseactivity,orNPA,ofmanukahoney.In2008laboratoryanalysisidentifiedthepresenceofmethylglyoxal(MGO)inhoneyproducedfromLeptospernumspecies(150)(88)(58).MGOispresentinhighconcentrationsintheflowerofthemanukabush,andisproducedfromdihydroacetate(138).MGOhasbeenshowntohaveantimicrobialeffectsmediatedthroughthedownregulationofautolysin,anenzymeimportantforcellulardivisionandcellwallregulationingrampositivebacteria.Alterationsinthecellwallregulationcauseafailureofcelldivisionanddysregulationofthecellwall(151).Ingram-negativebacteria,MGOseemstoaffecttheexpressionofgenesthatregulatecellwallstability.Theoutcomeiscelllysis(152)(153).Duringstorage,concentrationsofdihydroxyacetateslowlydecreasewhilstconcentrationsofMGOincrease.Thisactivityisusedtodeterminetheuniquemanukafactor,orUMF,ofthehoney(88)andaimstoaccuratelyreflecttheantimicrobialactivityofthespecificbatchofhoney(154).Theratingcomparestheantimicrobialactivityofabatchofhoneyagainstthatofastandardantisepticagent(phenol)againstStaphylococcusaureus(88)(155)(156).UMF-4hasminimalantimicrobialeffect,UMF5-9hasasmallantimicrobialeffectbutnotatlevelsthatareconsideredtherapeuticwhilstUMF-10-15isconsideredclinicallyuseful,andUMF16-30hasexcellentantimicrobialefficacy(157).However,manukahoney’santimicrobialeffectsarenotsimplyduetothepresenceofMGO.ThereisnotalinearcorrelationbetweenMGOconcentrationsandantibacterialeffects(154).Thereislikelytobeotherundiscoveredbioactiveagentscontributingtotheantimicrobialactivityofmanukahoney,anditislikelythatthesecompoundsactsynergisticallytocreateasuperiorproduct.(158)(159).Itisimportanttonote

theUMFratingonlyreflectstheantimicrobialactionsofmanukahoneybearsnoreflectiononitspotentanti-inflammatoryproperties.Biofilmsinequinewoundsarecommonandhavesimilarbacterialdiversitytothosefoundinchronichumanwounds(160).Experimentalmodelshavesuggestedthisisparticularlyaprobleminthedistallimbofthehorse(51).Manukahoneyhasbeenshowntobemoreeffectivethangenerichoney,inpreventingandeliminatingbiofilmsproducedbyStaphylococcusaureus(161).IthasbeenshowntonotonlyimpairPseudomonassppbiofilms,buttocompletelyeliminateinfectionswhenappliedatconcentrationsof64%and80%honey.Honeyistypicallyappliedatconcentrationsof100%,butclinicallywoundexudatewilldilutethehoneytolowerconcentrationsinthewound(162).Itwouldappearthatconcentrationsofmanukahoneytoeliminateestablishedbiofilmsarehigherthanthatrequiredtopreventbiofilms(161).Furthermore,studieshaveshownmethylglyoxal,isolatedfrommanukahoneyandusedaloneatconcentrationsequivalenttothatfoundinmanukahoneyisineffectiveagainstPseudomonassppbiofilms(145).Thishighlightsthatmethylglyoxalactssynergisticallywithotherbioactivecomponentsinmanukahoneytodeliverantibacterialandanti-biofilmeffects(145)(161)..Manukahoneyalsohastheabilitytomodulatewoundhealingdirectlyandhasbothantiandpro-inflammatoryeffects(57)deliveredbyregulatingtheinflammatoryresponseinthewoundandenhancingtheproductionofcytokinesthatregulatethetissueproliferationphaseofhealing(57).Ithasbeenspeculatedthatmanukahoneydoesnotsimplyjustupordownregulateinflammation,butratherenhancestheinflammatoryresponseintimesofalow-gradeinflammatorystate.Converselyduringahigh-gradeinflammatoryresponse,suchasduringanactiveinfection,itcanbedownregulated(149).Toll-likereceptor4(TLR-4)isanintegralpartofthisprocess.TLRshaveimportantrolesinthesignallingandultimateoutcomeoftheinnateimmunesystemastheyformagroupofpatternrecognitionreceptors(PRRs).Theyrecognisemicrobialmolecularpatternsandthensignal,vianuclearfactorκβ(NF-κβ),interferonregulatoryfactor(IRF)ormitogenactivatedprotein(MAP)kinases,toguidetheimmuneresponse(163).Activatedmonocytes,viatheNF-κβpathway,produceanarrayofcytokines(149).TheseincludeIL-1β,IL-6andTNF-α,allofwhichareimportantpro-inflammatorycytokines(164)andhavebeendemonstratedtobecriticalinresolvingwoundinfectionsandstimulatingrepair(142).Previousstudieshavespeculatedthathoneycontaminatedwithmicrobesmaybepro-inflammatory(165),asthepresenceofmicrobesandtheirendotoxinshaveastimulatoryeffect.Contaminationisplausibleviathehoneybeeitselforrawnectar(166).However,amorerecentstudyhasdemonstratedthatthereisaspecificbioactiveisolatecontainedwithinhoneythathasdirectstimulatoryeffectsontheTLR-4receptor.Thiscomponenthasbeenisolatedbasedonmolecularweight,andisa5.8kDamoiety(166).Furtherstudiesarecontinuingtocharacterizethisisolate.Importantly,thisstudyfoundnoassociationbetweenUMFandtheinflammationmodulatingeffectofmanukahoney(166).OtherstudieshavefoundchangesinIL-8production,amajoranti-inflammatorycytokine(1),invitro(167)(168)Furthertothese

recentdiscoveries,manukahoneyhasbeenfoundtoinhibittheexpressionofCOX-2andalsoreducetheproductionoffreeoxygenradicals(169)(170).Additionalresearchisrequiredinthisareatocompletelyclarifytheanti-inflammatorymechanismsofmanukahoney.TeaTree(Melaleuca)honeyRecently,researchersinAustraliahavediscoveredthathoneyderivedfromtheTeaTreehashealingproperties.TheAustralianTeaTree,orMelaleucaalternifolia,isusedtoproduceTeaTreeoilandisfoundonthenortheastcoastofNewSouthWales(171).TeaTreeoilhasawell-establishedscientificbackgroundforuseasamedicinalagent(172)(173).However,verylittlepublishedresearchontheuseofhoneyfromtheAustralianTeaTreeasamedicinalagent.OneprojectcommissionedbyaninvestmentgroupandconductedbyresearchersatSouthernCrossUniversityhasindicatedthatMelaleucahoneyhasstrongandimmediateanti-oxidantandanti-microbialproperties.Thisprojectconcludedthattheantimicrobialeffectswereimmediateanddidnotoccurinthepost-harveststage,asitdoeswithmanukahoney.Limitedspecificdetailsofthefindingsarecurrentlyavailable(174)andthisconfirmsfurtherresearchiswarranted.2.6ModelsofwoundhealinginthehorseModelsofwoundhealinginthehorsehavehistoricallyinvolvedsurgicallycreatedfullthicknesswoundsonthedorsomedialaspectofthemetacarpusormetatarsus.Thesestudieshavecreatedwounds,varyingbetween2cmx2cm,2.5cmx1.5cmand2.5cmx2.5cminsize,undershort-termintravenousanaesthesia.Thesemodelshaveevolvedovertimewiththeunderstandingofwoundhealinginthehorse.Initiallylimbswerekeptbandaged,forthepurposesofcontinuingcontactwithtopicalagents,whichresultedonaprotractedbandagingperiod(175).Overtime,withexpandingknowledgeofwoundhealing,bandagingbecameactivelydiscouragedpastday12afterwounding(44).Thislatterstudythatdemonstratedthebenefitsoflimitingbandagingusedanon-contaminatedwoundmodelwheresurgicallycreated2.5cmx2.5cmwoundsonthelowerlimbwereeitherbandagedorleftunbandaged.Bandagedlimbswerechangedregularlyuntilhealingwascompleted.Unbandagedlimbshadalightbandageplacedforthefirst24hoursforthepurposesofhaemostasis,butwerethenleftunbandagedforthedurationofthestudy.Itwasfoundthatbandagingthewoundssignificantlyprolongedthephaseofretraction,andthatabandagedwoundrequiredregularinterventionforthecontrolofexuberantgranulationtissue.Incomparison,unbandagedwoundshealeduneventfullywithnointervention.Totaldaystohealingremainedcomparablebetweenthetwogroups,butitisvalidtoassumethattherepeatedinterventionfortheexuberantgranulationtissueinbandagedlimbshastenedthehealingprocessandaddedanotherleveloftreatment.Hadnointerventionoccurred,itislikelythathealingwouldhavebeendelayed(44).Followingthesefindings,modelschangedtolimitbandagingtotheearlyphasesofthetrialsanddiscouragedbandagedbeyondday12,approximatelythepointatwhichgranulationtissuebedsstarttobecomeexuberant(44)(176).

Initialmodelsalsocreatedwoundsasepticallyundersurgicalconditions(44)(177).However,theclinicalrelevanceofthesemodelswasdebatableastheyfailedtoreplicatethetypicalclinicalsituationofgrosscontaminationoflowerlimbwounds.Thisledtothedevelopmentofcontaminatedwoundmodels,wherefreshfaeceswereappliedtothesurgicallycreatedwoundsforthefirst24hoursafterwounding(176).Instudiesutilisingcontaminatedmodels,thewoundsexperiencedatransientincreaseinbothaerobicandanaerobicbacterialcounts.Thisincreaseinbacterialcountdidnotresultinaclinicalinfection.Further,thesestudieschangedthepatternofhealingcomparedtoanuncontaminatedwound.Contaminatedwoundscontractedmoreandhealedfasterthanuncontaminatedwounds.Theauthorsspeculatedthatcontaminationofawoundimprovesandstimulatesthequalityoftheearlyinflammatoryphasethatistypicallysuboptimalinwoundsofthedistallimbofthehorse.Manukahoneyhasbeenwellstudiedinsecondintentionhealingofwoundsofthedistallimbofthehorse,usingcontaminatedanduncontaminatedwoundmodels.Incontaminatedmodels,manukahoneyhasbeendemonstratedtolimittheinitialphaseofwoundretraction,althoughsimilarbutlesspronouncedeffectsareseeninuncontaminatedmodels(176)(15).WhileUMF20manukahoneyhaswellestablishedantibacterialproperties,severalstudiesinthehorsehavebeenunabletodemonstrateadirecteffectonbacterialcounts.However,moststudiessuggesttheprimarymodeofactionofmanukahoneyonwoundhealingisthroughitsantibacterialproperties(20).ManukahoneyisalsoreportedtohavedirectinflammatorymodulatingeffectsmediatedthroughtheactionofToll-likereceptor4onneutrophils(20).Ultimately,theoveralltheactionofUMF20manukahoneyappearstopromoteavisiblyandhistologically,morematurebedofgranulationtissueearlierinthehealingprocess.Itwouldappearthebeneficialeffectsofmanukahoneyoccurintheinitial21daysfollowingwounding(176).FurtherstudiescomparedthewoundhealingpropertiesofUMF20manukahoneywitha66%UMF20manukahoneygelcomposite,andcontrolwoundsincontaminatedanduncontaminatedmodels.TheUMF20gelwasmadebycombiningthehoneywithawaterbasedgellingagenttoimprovecontactwiththewound..Theresultsshowedthat66%UMF20manukahoneygelwasaseffectiveaspureUMF20manukahoneyinpromotingsecondintentionwoundhealing(60).Consistentwithpreviousstudies,thisstudyconcludedthatmostofthebenefitsofmanukahoneywerefoundinlimitingwoundretractionintheinitialperiodafterwounding,buttherewasasmallbeneficialeffectonwoundstreatedwithmanukahoneygelthroughouthealing.Theauthorssuggestedtheremaybeclinicallybeneficialeffectsofapplying66%UMF20gelthroughouthealinginlarge,heavilytraumatisedwoundsthatoftenconfrontpractitionersinthefield.Dilutinghoneywithawater-basedgelreducestheneedforbandagestomaintaincontactbetweentheactivetopicalagentandthewound,reduceswastageandtheriskofexuberantgranulationtissuefromprolongedbandaging.Further,theabilitytodilutethehigherratedUMFmanukahoneymayreducethecostoftreatmenttotheclient.

Thenextstageofinvestigationwentontoquantifyantibacterialeffectsandimprovementsinsecondintentionwoundhealingfollowingapplicationofmanukahoney(20).A66%compositegelofUMF20manukahoneywasused,basedontheprevioustrials.Thisstudyfailedtodemonstrateasignificantreductioninbacterialcountsafterapplicationofhoneybutdidconfirmthatcontaminationwithfaecesresultedinhigherinflammatoryscores.Theauthorssuggestedtheshort-termapplicationoffaecesasawoundhealingmodelisinsufficienttocreatecontaminationandprovidesamodelmorerepresentativeofaclinicalsituationandsuggestedthatthecomplexityofthestudydesignmayhavelimiteddetectionofanti-bacterialeffects.Bothwoundstreatedwithhoneyandthosecontaminatedwithbacteriademonstratedareductionininflammatoryscores,andcontaminatedwoundshadlowerinflammatoryscoresthanuncontaminatedwounds.Histologicallytherewasimprovedangiogenesis,fibrosisandcollagenorganisationinmanukahoneytreatedwounds(20).However,manukahoneyhadnoeffectonthetemporalexpressionofTGF-B1orTGF-B3(20)(19).MoststudieshaveinvestigateddilutedorpureUMF20manukahoneybasedontheknowledgethathigherUMFratedhoneyhadmorepotentantibacterialpropertiesandthiswaslikelytobeassociatedwithimprovedwoundhealing.However,UMF20isexpensivecomparedtolowerUMFmanukahoneyandgenerichoney(178).FurthermoreitislikelythatthepotentantibacterialpropertiesofhigherUMFmanukahoneyisnotalwaysnecessaryinthetreatmentofsomewoundswherethemoregenericpropertiesofhoneymaybesufficient.Onthisbasis,astudyusinganuncontaminatedequinedistallimbwoundhealingmodelwasperformedcomparingUMF20manukahoneytoUMF5manukahoneyandastoreboughttablehoney.Thisstudyelectedtouseanon-contaminatedmodeltoreducetheantibacterialeffectsofUMF20ofmanukahoneyandevaluatethemoregenericeffectsofhoneyandthereportedinflammatorymodulatingeffectsofmanukahoney.InthisstudyUMF20manukahoneyshowedonlymodestimprovementsinoveralltimetohealing.Whencomparedtotheeffectsfoundincontaminatedmodelsofequinewoundhealing,thisimprovementwassmallsuggestingthattheantibacterialeffectsofmanukahoneyareimportantinwoundhealing.(20).Therewerenosignificantdifferencesinwoundhealingvariablesbetweengenerichoney,UMF5andsalinetreatedcontrolwounds.Mostgenericstoreboughthoneyundergoesheattreatmentduringprocessingwhichreducestheactivityofglucoseoxidase.Furthermore,prolongedstoragealsoseesareductioninglucoseoxidase(137).Basedontheresultsofthisstudygenericstoreboughthoneycannotberelieduponasatherapeuticinsecondintentionhealing.WhilethetherewasnosignificantdifferencesinwoundhealingvariablesbetweenwoundstreatedwithUMF5manukahoneyandthosetreatedwithgenerichoneyorsalinetreatedcontrolstherewasatrendtowardsimprovedhealing.UMF5manukahoneyissuggestedtohavelittleornoantibacterialactivitybutmanukahoneydoeshavetheabilitytomodulatetheinflammatoryresponseandwhiletheantibacterialactivityofmanukahoneyisratedastheUMF,theanti-inflammatoryactivityisnotrated.ItispossiblethetrendtowardsimprovedwoundhealingvariablesseenbetweenUMF5manukahoneyandthoseof

generichoneyandsalinetreatedcontrols,atleastinpart,reflecttheanti-inflammatorypropertiesofmanukahoney.WhilethestudyconcludedthatUMF20showedimprovementinwoundhealingtheauthorsconcludedthathistologywouldbehelpfultodistinguishwhetheranydifferencesinwoundhealingbetweentreatmentgroupsinthisstudyandcomparethesetopreviousstudies.Inallthesestudieswoundswerephotographeddigitallyonceweekly.Animageanalysissoftwareprogram(ImageJ)wasutilisedtoanalysethephotographsbeforestatisticalanalysisoccurred.Thisprovedtobeareliablemeansofmonitoringwoundhealing.(20)(176)(60)(178).Thecontaminatedwoundmodel,withlimitedbandaging,hasprovedtobeareliableproducerofstatisticallysignificantresults.

CHAPTER3:APILOTSTUDYINTOTHEEFFECTSOFTOPICALTREATMENTOFCANNABIDOLONSECONDINTENTIONHEALINGOFEQUINEDISTALLIMBWOUNDS3.1IntroductionThepurposeofthisstudywastoinvestigatethepotentialforCBDextracttomodulatesecondintentionhealingofdistallimbwoundsinhorses.Specifically,theaimwastodetermineifa1%CBDextractcombinedwithUMF5manukahoneyasacarrierwoulddeliverabeneficialeffectonsecondintentionwoundhealinginacontaminated,distallimb,woundmodelinhorses.UsingUMF5manukahoneyasthecarrierintheCBDpreparationwasattractivetoinvestigatethepossibilityofanysynergismbetweenmanukahoneyandcannabidiolthatwouldofferanovelnaturalproductforuseinequinedistallimbwounds.ThehypothesiswasthatthecombinationofUMF5manukahoneyandcannabidiolwouldimprovesecondintentionwoundhealingvariablesincontaminated,equinedistallimbwounds

3.2Materialsandmethods

SixadultStandardbredhorses(3geldings,3maresagedbetween3and10years)withoutevidenceofwoundsorscarsontheirforelimbswereusedinthestudy.Theinstitution’sAnimalCareandEthicsCommitteeapprovedthestudy.

Oneweekpriortothesurgerythehorseswereweighed,identifiedwithanumberedcollarandtreatedwithananthelmintic.Theywerehousedinpairsingrassyardsforthedurationofthestudy.Lucernehaywasfedat2.5%bodyweightindividedfeedsdailyandfreeaccesstowaterwasavailablethroughoutthestudy.WoundcreationEachhorseunderwentaclinicalexaminationonthemorningofsurgery.Thedorsalaspectsofbothmetacarpiinallhorseswereclipped.A14Gcatheterwasplacedintheleftjugularvein,themorningofsurgery,afterasepticpreparationandlocalanaesthesiaofthesite.Eachhorsereceivedasingledoseofphenylbutazone(2.2mg/kgIV)andtetanusprophylaxispriortosurgery.Horsesweresedatedwithxylazinehydrochloride(1.1mg/kgIV)andanaesthesiawasinducedwithdiazepam(0.1mg/kgIV)andketamine(2.2mg/kg).Anaesthesiawasmaintainedusingtotalintravenousanaestheticagents(TripleDrip).

Allwoundswerecreatedonthesameday.Theskinoverthemetacarpiwasnotasepticallyprepared.Atotaloffivefullthicknessskinwounds(2.5cmx2.5cm)werecreatedonthelimbtoreceivethethreewoundswasrandomlyassignedbyblindlypullingthehorsenumberandthetreatmentprotocoloutofahat.Thewoundsweremademedialtotheextensortendonsandwereevenlyspacedbetweenthecarpusandthefetlock.Woundswerecreatedbymakingafullthicknessskinincisionaroundastandard,flexibletemplateusingascalpel,andtheskinwasremovedwithsurgicalscissors.Twogramsoffreshfaeces

collectedfromahorsethatwasnotincludedinthestudywasplacedoneachwound,andeachwoundwasindividuallybandaged.Thebandageconsistedofalowadherent,absorbentdressingcuttosizetopreventcrosscontamination(Melolin,SmithandNephewMedicalLtd,HullEngland)coveredwithasoftgauzeroll(EasifixK,BSNMedical,Hamburg,Germany)andcompressedunderanelasticadhesivetape(TensoplastVet,BSNMedical,Hamburg,Germany).Thehorseswereallowedtorecoverunassisted.

WoundtreatmentsBandageswereremoved24hafterwoundcreationandthefaecesrinsedoffthewoundsusingsterilesaline.Thewoundsonthelegwithtwowoundsweretreateddailywitheither2mlofUMF5manukahoney(carrier),(ComvitaAustraliaPtyLtd,Brisbane,Australia)or1mlof1%CBDextract(equivalentof10mgCBD)in2mlofUMF5manukahoney(CBD).Thewoundsonthelegwith3woundsweretreatedwith2mlof0.9%sterilesaline(saline),2mlofUMF20manukahoney(UMF20),(ComvitaAustraliaPtyLtd,Brisbane,Australia),or2mlUMF5manukahoney.The1%CBDextractwasmixedwithacarrier(UMF5manukahoney)toprovideasimilartreatmentvolumetooptimisecontactwiththewound.Thetreatmentallocationoneachlegwaspredeterminedsothattreatmentsoneachlegwereevenlydistributedacrossallwoundsandallhorses.Treatmentswererandomlyassignedbypullingthehorseidentificationandtheassignedtreatmentprotocolforeachlegrandomlyoutofahat.Aftertreatment,woundswereindividuallybandaged,asdescribed,topreventmixingoftreatments.Woundswerebandagedafterwoundcreationandforatotalof13daysandbandagedwerereplaceddaily.Treatmentwasapplieddailyfor12daysunderabandage.Onday13bandageswereremovedandwoundswereleftopenandtreateddailyforatotalof42days.WoundmeasurementsDigitalphotographsofthewoundwereobtainedondays1,7,14,21,28,35,and42afterwoundcreationandwoundsweremeasuredusinganimageanalysissoftware(ImageJ,USNationalInstitutesofHealth,Bethesda,USA).A2.5x2.5cmtemplate(6.25cm2)wasplacednexttothewoundasastandardreference.Theareaofthewoundswasmeasuredthreetimesandtheaveragecalculatedtoproduceasinglemeasureforeachwoundforeachmeasurementday.Theareaofthegranulationtissue,notincludingtheadvancingedgeofepitheliumoneachsideofthegranulationbed,wasmeasured.Overalltimetocompletehealingwasrecorded.Woundswereconsideredcompletelyhealedwhengranulationtissuewasnolongervisible.Overallrateofwoundhealing(cm2/day)wasdeterminedbydividingthewoundareaonday1bythetotaldaystocompletehealing.StatisticalanalysisAgenerallinearmodelwasusedtoanalysethedata,witheachhorsehavingmultiplemeasurementsovertimeafterwoundcreation.Themodelincludedfixedeffectsoftreatment(saline,carrier,UMF20,CBD),day(1,7,14,21,28,35,42),leg(left,right),positiononleg(top,middle,bottom)andtheinteractioninvolvingtreatmentandday.Arandomeffectwasenteredforeachhorseto

accountforcorrelationsbetweenmeasurementsmadeinthesamehorse.Residualsfromthefinalmodelwereinspectedtocheckmodelassumptions.Summaryoutcomemeasureswerederivedatthelevelofeachwoundforeachhorsetorepresenttotaldaystocompletehealing,andoverallhealingrate(changeinwoundareaperdayfromday1tocompletehealing).Separategenerallinearmodelswereusedtoanalysetheseoutcomesusingthesameapproachasdescribedabove.Significanteffectsinthemainmodelwerethensubjectedtopairwisecomparisonsofestimatedmarginalmeansasfollow-uptests,usingstandarderrortermsderivedfromthegenerallinearmodel.Allfollowuptestsassociatedwithsignificantunadjustedp-valueswerethenadjustedusingtheFalseDiscoveryRateapproachtocorrectforriskofelevatedtype1errorduetomultiplecomparisons.Theanalysiswasfirstrunwithwoundstreatedwiththecarrier(UMF5manukahoney)onseparatelegsincludedasseparatetreatments.Initiallythiswasrunwiththewoundstreatedwiththecarrieridentifiedasleftorright.TheanalysiswasthensubsequentlyrunwithwoundstreatedbythecarrierbeingidentifiedasipsilateralorcontralateraltothewoundtreatedwithCBD.TheseanalyseswereruntodetermineiftherewasanylocaleffectofCBDonhealing.Theseanalysesshowednodifferenceinmeanwoundareaonanytreatmentdaybetweenanyofthetreatmentgroupssothemeanwoundareasofallthecarrierwoundswerecombinedforthefinalanalysis.AnalysiswereperformedinSTATA(StataDataAnalysisandStatisticalSoftware,StateCorp,LP,CollegeStation,TX,US)usinganalphathresholdof0.05.3.2ResultsAllhorseswereclinicallyhealthythroughoutthetrial.Therewasnoevidenceoflamenessorswellingandnowoundsdevelopedexuberantgranulationtissue.Horse#4wasobservedtointerferewiththebandagecoveringonewoundandthewoundinquestionappearedsubjectivelylarger.Therewasnodifferenceinwoundareabetweenanyofthewoundonday1followingwoundcreationbeforetreatmentcommenced.Thegeneralpatternsofhealingweresimilaracrossalltreatmentgroups(Figure1).Therewasnodifferenceinthedailymeanwoundareabetweenanyofthetreatmentsonanyofthetreatmentdays.

Figure1:Plotofmeanwoundareaforeachgroupoverthe42daysofthestudy.Barsrepresent95%confidenceintervals.Therewasnochangeinwoundareaforanyofthewounds,irrespectiveoftreatment,betweendays1and7.Afterday7,thereweresignificantdifferencesinmeanwoundareaovertimewithineachtreatmentgroup.Thedailymeanwoundareasfordays14to42weresignificantlysmallerthanday1and7meansinalltreatmentgroups(P<0.05).Inalltreatmentgroups,dailymeanwoundareafordays21to42weresmallerthantheday14mean(P<0.05).Dailymeanwoundareafordays14to42weresignificantlysmallerthanthemeanwoundareaonday7(P<0.05)foralltreatmentgroups.Beyondday21therateofreductioninwoundareaappearedtoslowandcomparisonsofmeanwoundareawithinanyonetreatmentgroupproducedsignificantresultsinsometreatmentgroupsandnon-significantresultsinothertreatmentgroups.Therewasnodifferenceinthemean(±SE)overallhealingrate(cm2/day)betweenanyofthetreatmentgroups(saline:0.108±6.5;carrier:0.113±0.006;UMF20:0.100±0.008;CBD:0.111±0.008).Therewasnodifferenceinthemean(±SE)totaldaystohealingbetweenanyofthetreatmentgroups(saline:84.9±0.008;carrier:83.2±5.0;UMF20:88.5±6.8;CBD:84.4±6.7).Woundareawithhorse#4omittedHorse#4wasobservedtohaveinterferedwiththebandagecoveringonewoundandthewoundareameasurementsappearedtobeimpacted(Figure2).

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44Day

Saline CarrierUMF20 CBD

Figure2:Lineplotofrawwoundareameasurementsanddisplayingonelineperhorse.Thelineforhorse=#4islabelled.Woundareameasurementswereinspectedforstatisticalevidenceofoutliermeasurements.Therewasnoevidencetosupportidentificationofanymeasurementsinanygroupasbeingoutliers,includingthemeasurementsfromthehorsethatinterferedwiththebandages(Horse#4).Consequentlytheanalysiswiththefulldatasetwasconsideredthemostappropriateanalysis.Althoughpreviousstudynumbersweresimilartothisstudy,aretrospectivepoweranalysiswasperformedandthisconfirmedthatthesamplesizeofsixhorsesdidnotimpactontheresults.Increasingthesamplesizeto8or10orevenabovethatwouldnothavechangedtheresults–assumingthatadditionalhorsesandwoundsbehavedthesamewayasthe6horsesthatwereincludedinthestudy.3.4DiscussionTherehavebeenmanyproductsrecommendedtoenhancesecondintentionwoundhealinginhorses,buttherearelimitedoptionsavailablethathavebeenshowntoreliablyenhancehealing.Thisstudyaimedtoinvestigatetheeffectsoftopicalcannabadiol(CBD),withacarrierofmanukahoney,onsecondintentionwoundhealing.CBDisanovelagentwithanti-inflammatoryandanti-fibroticeffects(113)(114)showingearlypromiseasatreatmentforsecondintentionwoundhealing.Thesepropertiesaresimilartotheinflammatorymodulatingeffectsofmanukahoney.Bothproductshaveantimicrobialpropertiesandarenaturalcompoundssothecombinationwasconsideredacommerciallyattractiveandinnovativecombinationofproducts.Theactivecomponents,thetherapeuticeffectsandtheinteractionbetweensuchcomponentsinbothproductsarestillbeingdeterminedbutpotentialsynergismexistsbetweenvarioustherapeuticcompoundswithinthe

Horse= #4

0 10 20 30 40

TreatmentCarrier

Wound area

marijuanaplanthasbeendescribed,suggestingthatcompoundslikeCBDandTHCtogetherexertastrongereffectthantheindividualcomponentsusedalone(179)(180)(181).Thisistermedtheentourageeffect.ItisalsospeculatedthatCBDmayhaveanentourageeffectwithterpenesorterpenoids,howeverthereislittlepublisheddataavailable(182).Infact,somestudieshaveattemptedbutfailedtoestablishthiseffect(182).Regardlessterpenesarefoundinhoneys(183),and,ifthereisanentourageeffect,wehypothesisedtheremaybepotentialforasynergisticeffectbetweenhoneyandCBD.Asdiscussed,wehypothesizedthenovelcombinationofmanukahoneyandCBDwouldenhancehealingofwoundsbymodulatingtheearlyinflammatorystageofhealing.Therewerenodifferencesbetweenthemeasuredwoundhealingvariablesfromwoundstreatedwiththedifferenttreatmentregimensinthisstudy.ThiswasunexpectedbecauseaseriesofrecentstudieshaveshownUMF20manukahoneyconsistentlyimprovesmeasuredwoundhealingvariableswhenapplied,daily,tocontaminatedoruncontaminated,surgicallycreated,experimental,distallimbwoundslefttohealbysecondintentioninhorses,whencomparedtocontrolwounds(20)(60)(176)(178).Inthesestudies,woundstreatedwithUMF20manukahoneyconsistentlyretractedlessthanuntreatedwounds(176)(60).Theauthorsproposedthatthepresenceandefficiencyofmyofibroblastsinamaturegranulationbedinthefirst7daysafterwoundcreationintreatedwounds,limitswoundretractionwhencomparedtountreatedwounds(176)(60).WhilethemostobviousbenefitofapplicationofUMF20manukahoneyisseeninthefirst7daysafterwoundcreation,improvedoverallhealingtimeshavealsobeenreported(60)(178).Unfortunately,UMF20manukahoneyisexpensive,whichledustoconsiderthelowergradeUMF5manukahoneyasacarrier.PreviousreportssuggestUMF5manukahoneyisgenerallynotrecommendedfortherapeuticuseasanantibacterialagentbecausetheknownactiveconstituentMGOisfoundinsuchlowconcentrations(178)(57)(157).Nevertheless,UMF5manukahoneywasconsideredanattractivecarrierfortheCBDinthispreliminarystudybecausedifferentvarietalsofhoneycontainmanyunidentifiedbioactivecomponentsthat,whencombined,contributetotherapeuticbenefitsofthespecificvarietal,andevenbatch,ofaspecifichoneyvarietal(178)(157).Trademarkedmanukahoneyistheonlyhoneywherethereisanattempttoprovideareliablemeasureofbioactivitybutthisissolelywithrespecttothehoney’santimicrobialproperties(178)WhilemostofthebeneficialeffectsofmanukahoneyonwoundhealingarecurrentlyattributedtoMGO,evidencewouldsuggestthattheanti-microbialefficacyofMGOappearstorelyonthepresenceofotherknown,andmanyunknown,antimicrobialandanti-inflammatoryconstituentswithinthehoney,toreachoptimalefficacy(57)(161).Giventhis,wehypothesisedthat,intheabsenceofaregionaleffect,thehealingvariablesinwoundstreatedwith1%CBDinacarrierofUMF5manukahoneywouldhavebeenexpectedtobeimprovedwhencomparedtothewoundstreatedwithUMF5manukahoneyaloneinthatsameleg.Topicallyapplied1%CBDcombinedinacarrierofUMF5manukahoneywasexpectedtohaveresultedinanimprovementinwoundhealingvariableswhencomparedtoUMF5manukahoneyaloneorsalinegroups.ComparisoncouldthenbemadetothewoundstreatedwiththemoreexpensiveUMF20manukahoney.Thesamecontaminatedwoundhealingmodelwasused(176)(60).Themean

woundareaonday1beforetreatmentcommencedwasconsistentacrossalltreatmentgroups.Thiswasanticipatedandconsistentwiththeresultsofpreviousstudies(176)(60)(178).Themeanwoundareainalltreatmentgroupswasthenexpectedtoincreasebetweendays1today7(176)(60)(178).However,therewasnochangeinthemeanwoundareairrespectiveoftreatment.Specifically,woundstreatedwithsalinewereexpectedtoretractmorethanwoundstreatedwithUMF20manukahoney,(176)(60)buttheresultsofthecurrentstudyshowedthatthepatternthroughouthealingwasthesameforalltreatmentgroupswithnoapparenteffectoftreatment.Therearefewlikelyexplanationsfortheunexpectedpatternofhealing(20)(176)(60)(178).OnepossibilityisthattherewasasystemiceffectofthetopicalCBD.CBDissuggestedtohavepotentialanti-inflammatory,(184)(185)antioxidant(94)andanalgesicproperties(186)aswellaspotentantibacterialeffects(184)(85).ItwouldappearthattheactivationofCB2receptorsincellssuchaslymphocytes,monocytes,macrophages,fibroblastsandkeratinocytes,amongstothers,(187)(188)(189)(190)suppressinflammationbyreducingexpressionofvariouscytokinesincludingtrans-forminggrowthfactorβ(TGF-β),transforminggrowthfactorα,interleukin-1andinsulin-likegrowthfactor-1(191)Inmostspecies,TGF-βhasthebroadestspectrumofactivityinwoundhealingandisdirectlyassociatedwiththeregulationofthefibroticresponse.(192)(193).Inhorses,thedifferentisoformsofTGF-βinteracttoplayapivotalroleinorchestratingwoundhealing.(19)(194)(195).TGF-β1peakssoonafterwoundingwhileTGF-β3peaks7–9dayslaterandmodulatesthefibroticresponse(19)(194)(195).Whiletheprocessofsecond-intentionwoundhealinginthehorseiscomplexandnotcompletelyunderstood,theconcentrationsandtemporalexpressionpatternsofthesetwocytokineswithinthewoundappeartobeessentialforthenormalprogressionofwoundhealing,particularlyinwoundsonthedistallimb(19)(194)(195).Itisbelievedinhorses,andinparticularindistallimbwounds,animbalanceintheexpressionofTGFβ1and3cancauseaweakandprolongedinflammatoryresponsethatleadstodelayedwoundhealing(31).IfthereweresystemiceffectsofCBDinthisstudy,itwouldhavebeenexpectedtohaveasimilarinfluenceontheprogressofwoundhealinginallwounds.BasedonthereportedpropertiesofCBD,thepotentialeffectsoftopicalapplicationinacontaminatedequinewoundhealingmodelwouldbeexpectedtobeassociatedwithitsantimicrobialandanti-inflammatoryeffects.Thewoundhealingmodelusedinthecurrentstudywasoriginallydevelopedtotryandeffectivelyreplicatenaturallyoccurringwoundsratherthantheaseptic,surgicalmodelsusedinearlierstudies(176)(60)(196)(178).Thepurposeofapplyingfaeceswastoreplicatewoundcontamination.However,whilethishasbeenshowntoincreaseaerobicandanaerobiccountsonthewounds,bacterialcountsonlyremainelevatedfor24h(20).Nevertheless,theapplicationoffaecesfor24hhasbeenshowntobesufficienttoaffectthepatternofhealingcomparedtonon-contaminatedwounds(60).Woundstreatedwithfaecesretractfurther,buthealfasterthanuncontaminatedwounds(60).Thissuggeststhattheapplicationoffaecesfor24hmaystimulatetheinitialinflammatoryresponseinequinedistallimbwoundsandenhancethewoundhealingprocesswithoutthenegativeeffectsofpersistentoroverwhelminginfectionoftenassociatedwithnaturallyoccurringdistallimbwounds(60).IfCBDwastohaveasystemiceffectinthismodel,itisverylikelythereportedanti-inflammatoryandantimicrobialpropertieswouldhavereducedwoundretractioninallwoundsinthefirst7daysafterhealing,

whichisconsistentwiththeresultsreportedinthecurrentstudy.Whendesigningistrial,itwasconsideredtherecouldbesystemicandregionaleffectsofCBDfollowingtopicalapplication.ItisdifficulttocomparethisprotocoltootheranimalmodelsastheavailablemicestudieshavegenerallyutilisedsystemicdosesofCBD,withoutcombinationwithotherproducts.Forexample,onestudyuseddosesofbetween5mg/kgand10mg/kgviaintra-peritonealinjectioninrodents(112).Experimentsutilisingthesetopicalagentsarerare,andnonearecurrentlyavailableusingequinemodels.However,availableevidencesuggestedanysystemiceffectwaslikelytohavebeenverysmallandunlikelytoaffectwoundhealing.OnestudyinvestigatingthetransdermaleffectsofCBDappliedtopicallyfortreatmentofpainandinflammationinarodent,adjuvant-induced,monoarthritismodelin260–280gratsfounddailydosesof6.2and62.3mgCBDappliedtopicallyfor4dayswereeffective,whilelowerdoseswerenot(197).Whiledailydosesof0.6and3.1mgwerenottherapeuticallyeffective,theywereabsorbedsystemicallyandrevealedadoserelated,linear,pharmacokineticcorrelationwhenthe6.2mgdosewasincluded(197).Whenthehigherdoseof62.3mgwasincludedinthepharmokineticcorrelation,itdidnotfollowthislinearrelationship(197)Theauthorsproposedthattransdermalabsorptioncouldbecomesaturatedwhenhigherdosesandhighervolumesofmedicationwereapplied,particularlyifthesedoseswereappliedtoaconfinedarea(197).TheauthorsalsopostulatedthatapplicationofCBDtotheskinoverlyingtheaffectedjointwouldpotentiallyincreasethelocalCBDconcentrationsandenhancelocalefficacywithoutsystemicinvolvement(197).Inthecurrentequinewoundstudy,woundstreatedwithCBDreceived10mgofCBDtopicallydaily.Thiswasconsideredamodestdosethatwouldhavelocaleffectswithouttherapeutic,systemiceffects.However,thecurrentstudyappliedtheCBDtoopenwoundsratherthantransdermally,soitispossiblethatsystemicabsorptionoftheCBDthroughanopenwoundmayhavebeenhigherandlessrestrictedbyareainthisstudycomparedtotransdermalapplicationintherat.Nevertheless,givendosesof0.6and3.1mgin260–280gratsappliedtopicallytotheskinwereabsorbedsystemicallybutnottherapeuticallyeffective(197),itwasconsideredadoseof10mgina500kghorsewasunlikelytodeliversystemicconcentrationsthatwouldbetherapeuticallyeffectiveinthecurrentstudy.TherehavebeennostudiesinvestigatingCBDinhorsessoitispossibletheeffectsofCBDorthesensitivityofCBDreceptorsaredifferentinhorsesthanrodents(197).Whilethecurrentstudydesigndidnotanticipateasystemiceffect,thestudydesigndidtakeintoaccountthepossibilityoflocaleffectsofCBD,foranypotentialeffectsofUMF5asthecarrier,andforanyinteractionbetweenCBDandUMF5manukahoney.Alone,UMF5manukahoneywasconsideredunlikelytohaveanyeffectonwoundhealingbasedonapreviousstudythatshowedUMF5manukahoneyhadnoeffectonwoundhealingvariablesinanuncontaminatedwoundhealingmodelinhorses(178).Althoughpreviousstudynumbersweresimilartothisstudy,aretrospectivepoweranalysiswasperformedandthisconfirmedthatthesamplesizeofsixhorsesdidnotimpactontheresults.Increasingthesamplesizeto8or10orevenabovethatwouldnothavechangedtheresults–assumingthatadditionalhorsesandwoundsbehavedthesamewayasthe6horsesthatwereincludedinthestudy.Giventhis,weremainconfidentthatthecauseofthe

lackofresultsismostlikelyasystemiceffectofCBD.

CHAPTER4:ASTUDYINTOTHEEFFECTOFINCREMENTALINCREASESOFUMFRATINGSOFMANUKAHONEYANDMELALEUCAHONEYONSECONDINTENTIONHEALINGOFWOUNDSOFTHEDISTALLIMBOFTHEHORSE4.1IntroductionUMF20Manukahoneyhasbeenshowntohavepositiveeffectsonsecondintentionwoundhealinginthedistallimbofhorsescomparedtountreatedcontrolwounds,woundstreatedwithgenericstoreboughthoneyandwoundstreatedwithUMF5manukahoney(179).TheantimicrobialeffectsofManukahoneymaycontributetotheimprovementinwoundhealingvariableshoweverManukahoneyalsohasbeenshowntomodulatetheinitialinflammatoryresponsethroughactivationofToll-likereceptor4onwhitecells(157).ThehighertheUMFofmanukahoneythemorepotentaretheantimicrobialeffects(157).UMF5and10haveminimalantimicrobialactivitywhileUMF15and20havesuperiorantimicrobialeffects(57).Whileantimicrobialactivityofmanukahoneyisquantifiedagainsttheantisepticphenol,theanti-inflammatoryeffectsofanybatchofmanukahoneyisnotrated(147).Furthermoretheremaybeotherbioactiveconstituentsofmanukahoneythatarecurrentlyunknown.ThecostofManukahoneyincreaseswiththeincreasingUMFfactor.WhileahighUMFfactormaybemoreappropriateinheavilycontaminatedwoundsorwoundswithestablishedbiofilms,lowerUMFandlessexpensiveManukahoneymaybeeffectiveinenhancinghealinginlesscontaminatedwoundsthroughthegenericfeaturesofhoneyandthespecificanti-inflammatoryeffectsofmanukahoneymediatedthroughToll-likereceptor4orotherunknownbioactiveconstituents.MorerecentlyMelaleucahoney(TeaTree)hasbeenproposedtohaveantimicrobialeffectshoweverthetherapeuticeffectshavenotbeenquantifiedorstandardisedlikeManukahoneyandanybenefitsonwoundhealinghavenotbeenestablished.ThepurposeofthisstudywastoinvestigatewhethertherewereanydetectabledifferencesonthewoundhealingvariablesbetweenManukahoneywithdifferentUMFsinacontaminatedwoundhealingmodelofthedistallimbofhorses.FurthermorewewantedtocomparetheeffectofMelaleucahoneyonsecondintentionwoundhealingagainstManukahoneyusingthesamewoundmodel.WehypothesisedthatusingthiscontaminatedwoundhealingmodelthatthewoundhealingvariablesofwoundstreatedwithUMF15andUMF20ManukahoneywouldbesuperiortothosewoundstreatedwithUMF5andUMF10manukahoneyandthatthewoundhealingvariablesofwoundstreatedwithUMF5andUMF10Manukahoneywouldnotbedifferenttosalinetreatedcontrolwounds.Further,wehypothesisedthatMelaleucahoneywouldnotimprovethewoundhealingvariablesinthiswoundhealingmodelwhencomparedtosalinetreatedcontrolwounds.

4.1Materialsandmethods

NineadultmixedsexStandardbredhorseswithoutevidenceofwoundsorscarsontheirforelimbswereusedinthestudy.ThestudywasapprovedbytheinstitutionsAnimalCareandEthicsCommittee.

Oneweekpriortothesurgerythehorseswereweighed,identifiedwithanumberedcollarandtreatedwithananthelmintic.Theywerehousedinpairsingrassyardsforthedurationofthestudy.Lucernehaywasfedat2.5%bodyweightindividedfeedsdailyandfreeaccesstowaterwasavailablethroughoutthestudy.WoundcreationEachhorseunderwentaclinicalexaminationonthemorningofsurgery.Thedorsalaspectsofbothmetacarpiinallhorseswereclipped.A14Gcatheterwasplacedintheleftjugularvein,themorningofsurgery,afterasepticpreparationandlocalanaesthesiaofthesite.Eachhorsereceivedasingledoseofphenylbutazone(2.2mg/kgIV)andtetanusprophylaxispriortosurgery.Horsesweresedatedwithxylazinehydrochloride(1.1mg/kgIV)andanaesthesiawasinducedwithdiazepam(0.1mg/kgIV)andketamine(2.2mg/kg).Anaesthesiawasmaintainedusingtotalintravenousanaestheticagents(TripleDrip).

Allwoundswerecreatedonthesameday.Theskinoverthemetacarpiwasnotasepticallyprepared.Atotalofsixfullthicknessskinwounds(2.5cmx2.5cm).Thewoundsweremademedialtotheextensortendonsandwereevenlyspacedbetweenthecarpusandthefetlock.Woundswerecreatedbymakingafullthicknessskinincisionaroundastandard,flexibletemplateusingascalpel,andtheskinwasremovedwithsurgicalscissors.Twogramsoffreshfaecescollectedfromahorsethatwasnotincludedinthestudywasplacedoneachwound,andeachwoundwasindividuallybandaged.Thebandageconsistedofalowadherent,absorbentdressingcuttosizetopreventcrosscontamination(Melolin,SmithandNephewMedicalLtd,HullEngland)coveredwithasoftgauzeroll(EasifixK,BSNMedical,Hamburg,Germany)andcompressedunderanelasticadhesivetape(TensoplastVet,BSNMedical,Hamburg,Germany).Thehorseswereallowedtorecoverunassisted.

WoundtreatmentsBandageswereremoved24hafterwoundcreationandthefaecesrinsedoffthewoundsusingsterilesaline.Treatmentswere2mlofUMF5manukahoney,UMF10manukahoney,UMF15manukahoneyorUMF20manukahoney(ComvitaAustraliaPtyLtd,Brisbane,Australia),2mlteatreehoneyor2mlsterile0.9%sodiumchloride(saline).Thetreatmentallocationoneachlegwaspredeterminedsothattreatmentsoneachlegwereevenlydistributedacrossallwoundsandallhorses.Treatmentswererandomlyassignedbypullingthehorseidentificationandtheassignedtreatmentprotocolforeachlegrandomlyoutofahat.Aftertreatment,woundswereindividuallybandaged,asdescribed,

topreventmixingoftreatments.Woundswerebandagedafterwoundcreationandforatotalof13daysandbandagedwerereplaceddaily.Treatmentwasapplieddailyfor12daysunderabandage.Onday13bandageswereremovedandwoundswereleftopenandtreateddailyforatotalof42days.WoundmeasurementsDigitalphotographsofthewoundwereobtainedondays1,7,14,21,28,35,and42afterwoundcreationandwoundsweremeasuredusinganimageanalysissoftware(ImageJ,USNationalInstitutesofHealth,Bethesda,USA).A2.5x2.5cmtemplate(6.25cm2)wasplacednexttothewoundasastandardreference.Theareaofthewoundswasmeasuredthreetimesandtheaveragecalculatedtoproduceasinglemeasureforeachwoundforeachmeasurementday.Theareaofthegranulationtissue,notincludingtheadvancingedgeofepitheliumoneachsideofthegranulationbed,wasmeasured.Overalltimetocompletehealingwasrecorded.Woundswereconsideredcompletelyhealedwhengranulationtissuewasnolongervisible.Overallrateofwoundhealing(cm2/day)wasdeterminedbydividingthewoundareaonday1bythetotaldaystocompletehealing.4.2ResultsAllhorseswereclinicallyhealthythroughoutthetrial.Therewasnoevidenceoflamenessorswellingandnowoundsdevelopedexuberantgranulationtissue.Therewasnodifferenceinwoundareabetweenanyofthewoundsonday1followingwoundcreationbeforetreatmentcommenced.Thegeneralpatternsofhealingweresimilaracrossalltreatmentgroups(Figure3).Therewasnodifferenceinthedailymeanwoundareabetweenanyofthetreatmentsonanyofthetreatmentdays.

Figure3:Plotofmeanwoundareaforeachgroupoverthe42daysofthestudy.Barsrepresent95%confidenceintervals.Therewassignificantincreaseinwoundareaforallofthewounds,irrespectiveoftreatment,betweendays1and8.Therewasthenasignificantreductioninwoundareafromday8today15inallgroups.Woundareacontinuedtosignificantlyreduceateachsuccessivemeasurementdayinallgroupsthroughtoday28(p<0.05).Beyondthistherewasacontinuedreductioninwoundareathroughtotheendofthestudy(day42)butcomparisonswerenotallsignificant.Fromday28today35,woundareasignificantlyreducedintheUMF5grouponly(p=0.04)andinallothergroupsthechangefromday28today35wasnotsignificant(p>0.05).Fromday35to42thechangeinwoundareawasnotsignificantinanygroup(p>0.05).Therewasnosignificantdifferencebetweentreatmentgroupsforoverallhealingrateordaystocompletehealing.4.4DiscussionTheresultsofthisstudywerecompletelyunexpected.Usinganestablishedcontaminated,equinedistallimbwoundhealingmodel,therewerenodifferencesinthemeasuredwoundhealingvariablesbetweenanyofthetreatmentsevaluated,orbetweenanyofthetreatmentsandcontrolwounds.Infact,thegeneralpatternsofhealingweresimilaracrossalltreatmentgroups.Woundsallwentthroughaninitialperiodofretractionfollowedbyaprogressiveperiodofwoundcontractionthatischaracteristicofthiswoundhealingmodel(20)(60)(176)(178).Incontrast,previousstudiesusingthiswoundmodelhaveconsistentlyshownwoundstreatedwithUMF20manukahoneyretractlessandshowbettergrossandhistologicalhealingwhen

comparedtosalinetreatedcontrols(60)(176)(178).Beneficialeffectsonwoundhealinghavebeenshowninbothcontaminatedanduncontaminatedequinedistallimbwoundmodels,althoughtheeffectsaremoremodestusinganuncontaminatedmodel(20)(60)(176)(178).Acontaminatedwoundhealingmodelwasusedinthecurrentstudytobetteralignthemodeltoanaturallyoccurringwoundwherecontaminationiscommon,andtopotentiateanydifferencesinwoundhealingvariablesasaresultoftheantibacterialeffectsofdifferentUMFgradesofmanukahoney(176)(60)(196)(178).Inacontaminatedwoundmodel,freshhorsefaecesareappliedtoeachwound,underabandage,for24hoursafterwoundcreation.Thismodelhasbeenshowntoleadtoatransientincreaseinbacterialcountswithoutcausingovertclinicalsignsofwoundinfectionbut,atthesametime,itprovidessufficientcontaminationtoalterthepatternofhealingcomparedtosalinetreated,controlwounds(20).TheeffectsofUMF20manukahoneyonwoundhealingusingthismodelbeenattributedtotheenhancedcontractileactivityofmyofibroblastsinamorematurebedofgranulationtissue,particularlyinthefirstweekafterwounding(176)(60).WhilestudiesdemonstratemostofthebeneficialeffectsoftreatmentwithUMF20manukahoneyoccurinthefirst3weeksafterwoundcreation,therearealsoimprovementsintheoveralltimestohealing(60)(178).BasedontheconsistentbeneficialeffectsofUMF20manukahoneyonamodelofsecondintentionhealingofdistallimbwoundsinhorsesshowninpreviousstudies,itislikelythatacompromisedstudydesignwasresponsibleforthefailuretodemonstrateaneffectofanyofthetreatmentsinthecurrentstudy(20)(176)(60)(178).Giventhesimilaritiesinstudydesignbetweenpreviousstudiesandthecurrentstudy,therearelikelytoonlybeafewpossiblereasonsforthefailuretodemonstratetreatmenteffects.Broadlyspeaking,itispossible,aloneorinsomecombination,establishinganytreatmenteffectwaslimitedbythesizeofthewounds,thenumberofwoundsandthenumberofdifferenttreatmentsoneachlimb.Thesizeandproximityofwoundstooneanotherinthecurrentstudydesignmayhavehadeffectsontheforcesresponsibleforretractionandcontractionofadjacentwounds.Inpreviousstudiesusingasimilarmodel,woundnumbersweregenerallylimitedtooneortwowoundsperlimbofsimilarsizeusedinthecurrentstudy(176)(60).Intwostudiesthatusedalargernumberofwounds,woundsizewasmuchsmallerthanthecurrentstudyandwoundswereoffsetratherthancreatedinaverticallinearrow(20)(196).Inoneofthesestudiesonlygrowthfactorsandhistologicalscoresweremeasuredandwoundhealingvariableswerenotanalyzed(20).Inthesecondstudy,woundhealingvariablesweremeasuredandtheseweresignificantlyimprovedinwoundstreatedwithUMF20manukahoneyora66%UMF20manukahoneygel,whencomparedtocontrolwoundstreatedwithsalineorthewaterbasedgellingagent(196).However,whilethenumberandsizeofthewoundsweresimilartothecurrentstudy,thedifferencewasthattherewerefewertreatmentsbeingevaluatedintheearlierstudy(196).Only2treatments,UMF20manukahoneyanda66%manukahoneygelwereevaluatedandthesetreatmentswereappliedfortwodifferenttimeperiods(12daysorthroughouthealing).Socomparedtothecurrentstudy,thisearlierstudy

hadalesscomplexdesign,itwascomparingfewertreatmentsandthetreatmentswerebothbasedonaUMF20manukahoneyandshowntohavethesamebeneficialtreatmenteffectswhencomparedtocontrolwounds.Thestudydesigninthecurrentstudyevaluatedalargernumberoftreatmentsandthesetreatmentswerelikelytohavesubtledifferencesintherapeuticactivity.Thesefactors,combinedwiththecloseproximityofthewoundstoeachother,mayhavelimitedtheexpressionanydifferencesbetweentreatmentsonwoundhealingvariables.Specifically,thetherapeuticbenefitsofthedifferentUMFmanukahoneysmayhavebeeninsufficienttoovercomeanyforcesaffectingtheretractionandcontractionofadjacentwounds.Whileitisalsopossibleovertcrosscontaminationofwoundsduringtreatmentcouldhaveoccurred,thiswasnotafeatureinpreviousstudiesusingthesamemodelandwasunlikelytohavebeenafactorinthisstudy.Itismorelikelythatdifferencesintreatmentonadjacentwounds,particularlywithUMFmanukahoneywithsimilartherapeuticactivity,wasnotdetectedinamorecomplexandambitiousstudydesignwithtoomanydifferenttreatments.Astudydesignusinglesswoundsandlesstreatmentsmayhavebeenmoreappropriate.Manukahoneyistheonlyhoneyvarietalwherethereisanattempttoprovideareliableandrepeatablemeasureofbioactivity.TheantibacterialactivityofeachbatchoftrademarkedmanukahoneyisstandardisedagainsttheantisepticphenoltoproduceaUMF.WhiletheUMFofmanukahoneyhasbeenlargelyattributedtothebioactivityofMGO,andtheconcentrationsofMGOinanybatchofhoneyhavebeenbroadlycorrelatedwiththeUMF,reportshavesuggestedthatothertherearemanyotherknownandunknownbioactiveagentsinmanukahoneywhichcontribute,asawhole,toitsefficacy(57)(161).So,whileitappearstheantibacterialpropertiesofmanukahoneycontributetothebeneficialeffectsonwoundhealing,theprecisemodeofactionofmanukahoneyonsecondintentionwoundhealinginthehorseremainsunclear.CurrentlythereislittlepublishedresearchaboutwhetherdifferentUMFmanukahoney,andconsequentlydifferentantibacterialefficacy,isdirectlycorrelatedwithwoundhealing.ArecentstudyusinganuncontaminateddistallimbwoundhealingmodelinhorsesshowedwoundstreatedwithUMF20honeyshowedsuperiorhealingtowoundstreatedwithUMF5manukahoneyorgenericstore-boughthoneybutthisstudydidnotestablishwhetherthisdifferencewasrelatedtotheantibacterialeffectsofmanukahoney,otherfactors,oracombinationofboth(178).ManukahoneywithaUMFfactorof5or10isreportedtohavelittleornoantibacterialactivityandisnotrecommendedfortherapeuticuseasanantimicrobialagent,whiletheefficacyofmanukahoneyagainstmanyoftheantibioticresistantbacteria,andestablishedbiofilms,increaseswithanincreasingUMF(157)(158)(198).TheaimofthecurrentstudywastoinvestigatewhethertheantibacterialefficacyofdifferentUMFmanukahoneywouldaffectwoundhealingvariablesdifferently.OurhypothesiswasthattherewouldbenoeffectofUMF5andUMF10manukahoneywhileUMF15andUMF20wouldimprovewoundhealingvariables.However,theresultsofthestudyfailedtoshowanyeffectoftreatment.ItispossiblethatthebacterialloadanddiversitycreatedusingthismodelwasnotsufficienttodemonstratesubtledifferencesbetweentheantimicrobialspectrumofthedifferentUMFfactors.However,previousstudiesthathavedemonstratedclearandconsistentimprovementsinwoundhealingvariablesbetweenwoundstreatedwithUMF20andsalinetreatedcontrolswouldsuggestthatthetransientand

modestbacterialcontaminationcharacteristicofthiscontaminatewoundmodelisunlikelytohave,alone,contributedtothefailuretoestablishdifferences.Otherfactorsthatmayhaveimpactedonthecurrentstudyarelesslikelytohavebeenresponsibleforthefailuretodemonstrateanytreatmenteffects.Thebrandofmanukahoneyusedinthecurrentstudywasdifferenttothebrandusedinpreviousstudies,soitispossiblethetwobrandshaddifferenteffectsonwoundhealing.However,themanufacturersproducingeachbrandoftrademarked,manukahoneyverifytheUMFoftheirhoneythroughinhouseandindependenttesting.Nevertheless,thereislittleresearchpublishedthatspecificallyaddressesfactorsthatmightreducethebioactivityofmanukahoney.Forexample,generichoneyisoftenheattreatedandstoredforlongperiodsthatisknowntoreduceitsbioactivity(137)(178)(57).IncontrasttheconversionofdihydroxyacetoneintoMGOinmanukahoneyincreasestoapproximately30%followingstorage.Nevertheless,itispossiblethatthebatchesofhoneyusedinthisexperimentwerecompromisedinsomewayPreviousstudiesusingthismodelhaveusedadultStandardbredgeldingsandmaresofmixedages.Welimitedtheupperageofthestudygroupto11yearstominimizeanysuggestedageeffectsordiseasessuchasCushing’sdisease,onhealing(199)(200).NoneofthehorsesinthestudyhadclinicalsignsofCushing’sdiseaseandtheagesofthehorsesweresimilartopreviousstudiessoitisunlikelyagefactoredintotheresultsofthisstudy.TheadditionofMelaleucahoneytothisstudyaddedanotherlayerofcomplexitytothestudydesign.However,Melaleucaalternifolia(AustralianTeatree)andLeptospermumscoparium(Manukabush)aredifferentspeciesoftheMyrtaceae(Myrtle)family.TeatreeoiliswellestablishedasatherapeuticagentbutthereiscurrentlylimitedevidencesupportingtheuseofTeatreeoilorhoneyforwoundhealing(172).Melaleucahoneyissuggestedtohavehighconcentrationsofanti-oxidantsaswellasantimicrobialactivity(174).Antioxidantspreventfreeradicalformation,scavengefreeradicalsorpromotethebreakdownandmayplayaroleinwoundhealingbypreventingfreeradicaldamage(201).Lowlevelsofantioxidantsinwoundshavebeenlinkedwithdelayedhealinginanimalmodels(202).ThestudylentitselftoevaluateMelaleucahoneybycomparingitwiththeknowneffectsofUMF20manukahoneyonwoundhealingonandtothepotentialeffectsofdifferentUMFmanukahoney.ThefailuretodemonstrateanytreatmenteffectsinthecurrentstudywouldsuggestfurtherstudieswillberequiredtoproperlyevaluateanypotentialeffectsofMelaleucahoneyonwoundhealing.Wedonotbelieveitispossibletodrawanyreliableconclusionsfromthecurrentstudy.Previousandsimilarstudydesignsusing6-9horseshavebeenshowntocapturestatisticaldifferencesbetweentreatmentgroups.Thefailuretodemonstratedifferencesinthecurrentstudy,inparticularestablishedeffectsofUMF20manukahoney,onsecondintentionwoundhealingvariables,wouldsuggestaflawinthecurrentstudydesignratherthatalackoftreatmenteffect.Wewouldconsiderthatitislikelythestudydesignbecametoocomplicated.Inthisstudyweinvestigated5treatments,4ofwhichwereevaluatedtoinvestigatesubtledifferencesintheeffectsofantimicrobialefficacyalongtheUMFgrading

scale.Thecontaminatedwoundhealingmodelonlycreatesatransientincreaseinbacterialcountsand,incombinationwith6largewoundsperhorse,mayhavemadeitdifficulttoestablishanydifferencesbetweentreatments.Inretrospect,thestudywastooambitiousandsetouttoevaluatetoomanytreatmenteffects.Infuturestudies,keepingthedesignsimpleandlimitingthenumberoftreatmentsiswarranted.TheadditionofMelaleucahoneytothestudyaddedtothecomplexityandmayhavebeenbetterlefttoaseparatetrial.

CHAPTER5:CONCLUSIONSANDFUTUREDIRECTIONSEquinedistallimbwoundsthatarelefttohealbysecondaryintentioncontinuetoposeachallengeforownersandveterinarians.Thetaskcontinuestofindaproductthatwillconsistentlyenhancethehealingofsuchwounds.Inthispilotstudy,wewereunabletodemonstrateaneffectofthenovelcombinationof1%CBDinacarrierofUMF5manukahoneyappliedtopicallyfor42daysonwoundhealingvariablesinasecondintentionhealingcontaminatedequinedistallimbwoundmodel.Despitepotentialissueswithstudydesign,thelackofanystatisticaldifferencesbetweenanytreatmentgroupswassurprisingtotheresearchersgiventhepreviousreliabilityofsimilarmodelsinproducingstatisticallysignificantresults.Althoughthispilotstudyintothecombinationofmanukahoneyandcannabidiolonthesecondintentionhealingofequinedistallimbwoundsdidnotproduceaneffect,wedonotnecessarilyconsiderthisafailureofthecannabidiolresin.Tothecontrary,whileitispossibletheremayhavebeenotherunknownfactorsresponsiblefortheunexpectedresultsinthisstudy,asystemiceffectofCBDseemsmostlikelygiventhedeviationawayfromthenormalretractionphaseafterwounding.MorestudiesintotheeffectsofCBDonequinewoundhealingthataccommodatespotentialsystemiceffectsofCBDwouldneedtobedevelopedtovalidatethisassumption.However,anyfurtherstudieswouldbecomplicatedbytheinabilitytouseeachhorseasitsowncontrol,soanystudywouldneedalargerstudypopulationtoaccountfordifferencesinwoundhealingbetweenhorsesandwouldaddanotherlayerofcomplexityandcosttotheexperimentalmodel.Thecombinationofmanukahoneyandcannabidiolstillremainsapotentiallyattractiveoptionandmay,infuturestudieswithrefinedmethodologies,provetoabetreatmentpanaceaforsecondintentionwoundhealingofthedistallimbofthehorse.Furthermore,althoughwefailedtoestablishdifferencesinhealingwithvaryingUniqueManukaFactorhoneygrades,previousstudieshavesuggestedthatusageofUMF20manukahoneyisstillencouragedoverotheroptions,includingUMF5manukahoneyandgenerichoney(178).OursuspicionremainsthattherecouldbeincreasingimprovementinhealingpatternsastheuniquemanukafactorincreasesincrementallybetweenUMF5andUMF20andevenabove.Thisparticularstudymayhavecreateditsowndownfallinattemptingtoresolvemultiplequestionsinonetrial.Wehypothesisethatthenumberofwoundsofthedistallimbmayhave,initself,createdaregionaleffectandobscuredanymeaningfuldata,reducinganydifferencesbetweenvaryingUMFtreatmentgroupstobelowstatisticallydetectablethresholds.Alternatively,issuessuchascrosscontaminationbetweengroupsmayhaveoccurredandchangedourresults,butweconsiderthesepotentialissuesunlikelygiventhereliablesuccessofthismodelinpreviousstudies.Regardless,addingintheTeaTreehoneyasatreatmentgroupmayhavecomplicatedthisstudyunnecessarily.Assuchtheresearchgroupfeelsthereisastrongneedforfurthertargetedresearchinthesefields,butwerecommendasimplermodelwithmorelimitedtreatmentgroups.WeremainencouragedtocontinueinvestigatingtheoutcomesofvaryingUniqueManukaFactorhoneysonsecondintentionhealingofwoundsofthedistallimbofthehorse

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[198]Wallace,A,Eady,S,Miles,M,Martin,H,McLachlan,A,Rodier,M,etal.DemonstratingthesafetyofmanukahoneyUMF20+inahumanclinicaltrialwithhealthyindividuals.BritJNutr2010;103(7):1023-1028

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[213]Bracamonte,S,Hendrick,J,Carmalt,J,Wilson,D.Exvivocomparisonofburstingstrengthofventralmedialandrightventralparamedianceliotomiesinhorses.VetSurg2013;42(4):468-472

[214]Bisogno,T,Hanus,L,dePetrocellis,L,Tchilibon,S,Ponde,D,Brande,I.Moleculartargetsforcannbidiolanditssyntheticanalogues:EffectonvanilloidVR1receptorsandonthecellularuptakeandenzymatichydrolysisofanandamide.BritJofPharmcol2001;134(4):845-852

[215]Eteraf-Oskouei,T,Najafi,M.Traditionalandmodernusesofnaturalhoneyinhumandiseases:areview.IranJBasicMedSci2013;16(7):731-742

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APPENDICESAppendix1:AnimalethicsANIMALRESEARCHETHICSCOMMITTEEAPPLICATIONFORMPleaseNote:ThisformwascreatedonlineinIRMAandtheinformationprovidedisrecordedintheUniversityResearchOfficedatabase.SignoffbyresearchersisprovidedonlineinIRMAandwillnotbedisplayedinthisdocument.ADMINISTRATIVEDETAILS

Title: Do the cannabinoids, cannabidiol (CBD) and tetrahydrocannabinol (THC),enhancesecondintentionhealingofcontaminateddistallimbwoundsinhorsesandaretheyaseffectiveasUMF20manukahoney.

ChiefInvestigator: ProfAndrewDartPrimaryFaculty/Department: Vet Science Faculty; Faculty of VeterinaryScience

Investigators: DartAndrew;TsangAlbert;

Grantslinked:ExternalAuthorities:

AdditionalInformation:Marijuana (Cannabis sativa) contains more than 60 biologically active chemicalagents thathavebeen shown tohavepotentantibacterial andanti-inflammatoryactivityaswellas theability to improveelementsofhealing inaratskinwoundhealingmodel.RecentstudiesbyourgrouphaveshownthatUMF20manukahoneyiseffectiveatstimulatingandimprovingtherateofwoundhealinginthedistallimbofhorses.Theaimofthisstudyistocomparewoundhealingvariablesfromwoundstreated with two active agents derived from marijuana, cannabidiol (CBD) andtetrahydrocannabinol(THC),UMF20manukahoneyanduntreatedcontrolwounds.

ANIMALNUMBERS

CountryJurisdiction InvasivenessLocation Classification1 Classification2 Common/strain AppliedRe-used

AustraliaNSW 4.Minor

surgerywithrecovery

DomesticmammalsHorses Standardbred 12 0

QUESTIONNAIRE

1 - <div align="left">Welcome to the University of Sydneyâ€™s Animal EthicsApplicationQuestionnaire.Pleasebeawarethatthereisalimitoffifteenminutestocompleteeachindividualquestion.Ifyouexceedthistimethenyouranswermaynotbesavedbythesystem.Werecommendthatyoupreparelonganswersoutsideof IRMA before pasting it back into the report questionnaire and/or save youranswers regularly.If you choose to edit a previous question, your responses tosubsequentquestionswillbedeleted.Therestorebuttoncanbeusedtorefillyoursubsequent answers if this happens.For further information on the applicationprocedure, please consult our <ahref="http://sydney.edu.au/research_support/ethics/animal/">website</a> oremailtheAnimalEthicsteamatanimal.ethics@sydney.edu.auIfyouexperienceanytechnicaldifficulties,pleasedonothesitatetocontactResearchSupportusingthedetailsbelow:T+61286278183Eresearch.support@sydney.edu.au</div>

Continue2-WhatistheChiefinvestigator'srelationshiptotheUniversityofSydney?Ifmorethan one applies, please select the option that is most relevant to this AECapplication.

Employee3-Pleaseindicatewhetherthisapplicationis fora:NewprotocolOrProjectwhichhas previously/simultaneously been submitted to this or another animal ethicscommittee

NewProtocol5-IsanyoftheworkforthisprotocolbeingundertakenataninstitutionotherthantheUniversityofSydney?No

7-DoestheprojectintendtouseSchedule8drugs?

Yes10-Pleaseselectallschedule8drugsthatyouintendtouse

Other11-Pleasespecifyanyotherschedule8drugsthatyouareusing(ifany)

Ketamine:RoutineuseattheveterinaryhospitalforanaesthesiaTetrahydrocannabinol:hasrecentlybeenrescheduledtoaschedule8drug.Itwillonlybeusedatlowdosetopicallyonthewounds.(CBDisschdule4)

Asveterinarianswearelicensedtouseschedule8drugs9-<divalign="left">Whattypeofapplicationisthis?Pleasenote,breedingprotocolsmust be submitted as a separate application to experimental protocols.Privatelyownedanimalsincludeanyanimalsthatare:-notownedbytheresearcher,and/or-are not 'wild' animals. Examples include:-pets (e.g. cats, dogs, horses)-animalsownedbyaprivateentity(e.g.Acommercialfarm,azoo)</div>

Experimental(non-wildlife)12-Doesthisprotocolinvolvethedevelopmentofnewveterinaryoragriculturalchemicals?Thisincludestheunapproved('off-label')useofanexistingregisteredveterinary/agricultural chemical product; or the use of an unregisteredveterinary/agriculturalproduct(includingnaturalproducts).

No13-Isthisprojectcommerciallysponsoredand/orsupported?

15-AreanimalsusedinthisprotocollocatedinNewSouthWales?Yes19-Youhaveindicatedthatanimalsusedinthisprotocolwillbe locatedinNewSouthWales.Pleasechoose themostappropriateoption todescribe theprimarypurposeoftheprotocol.

Research:healthandwelfare20-WildlifeResearch:YoumayrequireascientificlicencefromtheNSWNationalParks andWildlife Service. This permit is obtained after AEC approval. Furtherdetails are available <ahref="http://sydney.edu.au/research_support/ethics/animal/">here</a> on theanimalethicswebsite.IconfirmthatIwillobtainallrequiredpermitsandapprovalspriortoundertakinganyresearch

18-AreanimalsusedinthisprotocollocatedoutsideofNewSouthWales?No21-Brieflydescribetheaimsoftheprotocolinscientificterms(notmorethan500words).

Marijuana (Cannabis sativa) contains more than 60 biologically active chemicalagents thathavebeen shown tohavepotentantibacterial andanti-inflammatoryactivityaswellas theability to improveelementsofhealing inaratskinwoundhealingmodel.RecentstudiesbyourgrouphaveshownthatUMF20manukahoneyiseffectiveatstimulatingandimprovingtherateofwoundhealinginthedistallimbofhorses.Theaimofthisstudyistocomparewoundhealingvariablesfromwoundstreated with two active agents derived from marijuana, cannabidiol (CBD) and

tetrahydrocannabinol(THC),UMF20manukahoneyanduntreatedcontrolwounds.46- If theprotocolrepeatspreviouslyreportedexperiments,explainthereasonswhytheexperimentsneedtoberepeated.

No47-Whatspeciesofanimalwillbeusedinthisprotocolandwhy?

Equuscallabus:StandardbredhorsesHorsesareveryprone to traumaticandcontaminatedwoundsof thedistal limb.Theyareunique,withwoundhealingbeingmoreproblematicthaninotherspecies,includingponies.Woundsofthedistallimbsdonothealaswellasthoseofthebodyso there is a need for products that promote the healing of horse wounds,particularlythoseofthelowerleg.Manyproductsusedinotherspeciesdonothavethesameeffectinhorses.WehaveshownUMF20manukahoneypromoteslowerlimbwoundhealingindistallimbwoundsinhorses.Wehavedevelopedauniquecontaminated wound model which more closely replicates naturally createdwoundswithoutmany of the complications seen in naturally occurringwounds.Stadadrdbreds by virtue of their limited genetic diversity and temperament areidealforthismodelandhavebeenusedinallourpreviousstudiessoallowsomeintenrpetations and assumptions to be made between studies which reducenumbersofhorsesusedandrefinesthemodels.48-Pleaseprovideanexplanationofwhyanimalsareneededinthisprotocolandaclear description of the steps taken to consider and apply the 3Rs (Reduction,Refinement, Replacement). Please include a list of any potential alternatives toanimaluse,whetheranyof thesearebeingused in thisprotocol,and ifnot,whyalternativesareunsuitableforthisprotocol.Woundhealingisacomplexdynamicphysiologicalprocessthatistemporallyandspatiallyorchestratedandcanonlybestudiedinlivingtissues,Wehaveconductedmorethan5previousstudiesusingthismodelandwehavebeenabletoestablishthe numbers of horses needed to detect clinically relevant differences and haverefinedthemodeltousetheminimumnumberofhorsesandreduceanysideeffects.

49-Explainindetailhowyouhavedeterminedthenumberofanimalsnecessaryforthisprojectanddescribethewaysthatyouproposetominimisetheuseofanimals.Forexperimentalprotocols,includedetailsofstatisticalsignificance.Pleasedonotincludedetailsofmethodologyorimpactinthissection.Wehavefoundinpreviousexperiments6-8horsesaresufficienttodetectclinicallyrelevantdifferencesinwoundhealinginthismodel.Weaimtouse2groupsof6horses(12intotal).Thedoseofcannabinoidsweareusingtopicallyisconsideredtolowtohavesystemiceffectsbutitispossiblewemaygetlocaleffectsonadjacentwounds.Soweneed to test theCBDandTHC indifferenthorses.Ouraim isnotnecessarilytocompareTHCorCBDtoeachotherbuttocomparethemtoknowneffectivewoundhealingagent(UMF20manukahoney)andagainstasalinetreatedcontrolandthecarriervehiclefortheCBDandTHCgels.50 -Reuse:Does thisprotocol involve theuseofanyanimals thathavebeen thesubjectofpreviousresearch/teaching?

51-Willanyanimaltissuebeavailabletobesharedwithotherinvestigators?Ifso,pleasespecifywhichtissuescanbeshared.Ifanimaltissuescannotbeshared,pleaseoutlinethereasonsforthis.No.Theseanimalswillnotbesacrificed.Thisisaminimallyinvasivestudy.WehaveworkedwiththeAECpreviouslytohavearesearchherdofhorsesheretoconductminimally invasive studies which results in using less horses while providingotherwiseunwantedhorsesaqualityoflife.Dependingontheprojectsplannedforthefuturewemayelecttokeepthesehorsesandrequesttheabilitytousethemforotherstudiesorifwehavenofurtherstudiesonthehorizonputthembackthroughthesales.53-Sequenceofevents:Giveaclearstepbystepdescriptionofwhathappenstoanimals from the time you obtain/encounter them until the time the project iscompleted. Where possible, please attach a flow chart in the documents tab todescribewhatwill happen to animals and their tissues.Youmust indicatewhichprocedures will be performed in sequence to the same animal(s).<fontcolor="red">Where appropriate, SOPs may be used - please attach them to thedocuments tab.</font>You may click on the documents tab now, upload thedocument,thenreturnbacktothistab.1.Horseswillbeacclimatedandquarantinedfor2weeksafterarrival2.Horseswillthenbebroughtuptoyardsclosertotheworkingareasandhousedincompatiblepairsinadjacentyards

3.Horseswillbeweighed,wormedandvaccinatedfortetanus

4.Ajugularcatheterwillbeplacedonthedayofsurgery5.Phenylbutasone2.2mg/kgwillbegivenorally6.Anaesthesiawillbeinducedusingxylazine(1,1mg/kg)diazepam(0.1mg/kg)andketamine(2.2mg/kg)

7.Metacarpiwillbeclippedandasepticallyprepared8.Twofullthicknessskinwounds(2.5cmx2.5cm)willbemadeoneachmetacarpusevenly spaced between the carpus and fetlock dorsomedially adjacent to theextensortendon.9.Freshfaecescollectedfromanotherhorsenotinthestudywillbeplacedonthewoundstocoverthemandthelegwillbebandagedovernight.10.Thedayaftersurgerybandageswillberemovedandthefaecesrinsedoffwithsterilesaline.

11.Digitalphotographswillbetakenofeachwoundwithastandardisedtemplateadjacenttothewoundtocontrolformagnification12.Woundsononelegofall12horseswillbetreatedwith2mlUMFmanukahoneyor2mlofsterilesaline.13.Woundsontheotherlegof6horseswillbetreatedwith2mlofTHCgelorthecarrierforTHC,whileintheother6horseswoundswillbetreatedwith2mlCBDgelorthecarrier.14. Bandages will be reapplied and changed and the wounds treated with theassignedtreatmentfor12days.

15.Onday13bandageswillberemovedandthewoundleftopen.Dailytreatmentwiththeassignedmedicationwillcontinueuntilthewoundshavehealed.16.Woundswillbephotographedweeklyfor6weeksandthetotaltimetohealingwillberecorded.17.Woundswill bemeasured using a computer programwound size on day 1,wound size during the first 6 weeks of healing, total time to healing, and dailyhealingratewillberecordedandcomparedstatistically.54 - Impact: Identify all factors and procedures thatmay have an impact on ananimal's well-being. This may include handling, housing etc as well as specificexperimentalprocedures.Pleaserefertothechecklistavailablehereontheanimalethicswebsite, toensurealldetailshavebeenconsidered.Describeeachfactororproecedureandhowanyadverseimpactwillbeminimised.1.Horseswillbekeptasaherdandwheninseparateyardswillbeineyesightofeachother.2.Theywillbehandledandourexperienceisthattheyacclimatewelltothedailyroutines

3. Local anaestheticwill beused todesensitise the skin toplace the intravenouscatheter4.Horseswillbeanaesthetisedbyaveterinaryanaesthetistinapurposebuiltfaciiliy

5.Procedureswillbeperfromedunderanaesthesia6.Outexperienceisthatthesewoundsarenopainfulandhorsesarenotlameandlegsarenotswollen.

7.Apre-emptivedoseofphenylbutasoneisgivenpriortocreatingthewounds.8.Woundswillbecreatedbyanequinesurgeon9.Horseswillbesedatedifrequiredforanyofthebandagechangesorphotographysessionsiftheyareanxious

10Horsesarehousedandproceduresareperformedata24/7veterinaryfacility11.DrTsangandDrDartarelessthan15minutesawayandiftheyareunavailableornotcontactablethereisaveterinarianoncall24/7

55-Ifyouexpectanimalstodie(naturally/unassisted)duringthisprotocol,pleaseoutlinethepercentageofanimalsthatcouldbeaffected(i.e.theattritionrate)andexplainthelikelyreasonfortheanticipatedattritionrate.

Nodeathsareanticipated56-MonitoringPleaselistinvestigatorsresponsibleformonitoringoftheanimalsfor all of the following periods:-duringweekdays,-at night (if applicable)-duringweekends and holidays.-in an emergencyThis must include the methods andfrequencyofmonitoringoftheanimalspre,duringandpostprocedure.

DrDartandDrTsangwillmonitortheanimalsduringweekdaystwicedailyDrDartandDrTsangwillmonitor theanimalsduringweekendsandholidaysatleastoncedaily

DrDartandDrTsangwillbeavailableforemergencies

The hospital is a 24/7 facility with a veterinarian always on call and final yearstudentsonovernightwatch.Horsesarefedbyexperiencedstablehandsallyeararoundtwicedaily.Allhorsesinhsoptialarecontinuouslymonitoredandwalkedby.57-Whatisexpectedtohappentotheanimalsatthecompletionoftheprotocol?

Reused58-Specifythescientificendpointsofthisprojectandstatethereasonforselectingthese.

Theexperimentwillbeoverwhenthelastwoundheals.Weareevaluatingwoundhealingandneedtotaltimetohealingasavariable.59-Otherthanscientificendpoints,whatfactorsaffectingthewell-beingofanimalswill lead topre-mature terminationof theexperiment (i.e.whatare thehumaneendpoints)? Please include clinical signs that may or may not be related to theprotocol.Pleaseuploadamonitoringsheetintothedocumentstab.Youmayclickonthe documents tab now, upload the document, then return back to this tab. Astandard monitoring sheet outlining humane end-point criteria is available <ahref="http://sydney.edu.au/research_support/ethics/animal/">here</a> on theanimalethicswebsite.Thereisnotexpectedtobeanyreasontoterminatethestudyforanyofthehorses.Anyhorsethatgetsinjuredorsickwillbeassessedthoroughly.Ifanyhorsecannotcontinue for veterinary reasons it will be removed from the study. Any horserequiringtreatmentwithdrugsthatmayaffect thestudywillberemoved. In thecase of a catastrophic injury such as a fracture or an illness that offers a poorprognosishorseswillbehumanelydestroyed.60 - Incaseofplannedorunplanned(emergency)euthanasia,pleaseoutline thefollowing: -Howwill this be done? -Whowill perform euthanasia?-Howwill thenominatedpersonbecontacted?-Wherewilleuthanasiabecarriedout?-Howwilldeathbeconfirmed?-Howwillthecarcassbedisposedof?Ifthehorsecanbemoveditwillbeperformedinarecoverystalloutofsight.Ifitcannotbemoveditwillbeperformedinthepaddockwiththeotherhorsesremoved.WewilluseanoverdoseofpentobarbitoneIVDrDartorDrTsang-bothperformeuthanasiaregularlyaspartoftheirjob

DrDartandDrTsangarecontactable24/7byphoneAsabove

Abscenceofaheartbeatusingastethoscope

Dismemberdandplacedinmedicalwastebinsthatareprofessionallyremoved61-WillanimalsbehousedinanLASmanagedfacility?LASmanagedfacilitiesare:-Bosch- Brain andMind Research Institute- The Cattery- Charles Perkins Centre-Gunn Building- McMaster Annexe- Medical Foundation Building-Psychology/Badham-SchoolofMolecularBioscience

No62-HousingandManagementPleaseprovidefurtherdetailonwhereanimalswill

behoused/located.Pleaseincludethefollowing:-Describethetypeofhousingtobeprovided.-The maximum and minimum number of animals per cage/pen/tank.-Whattheanimalswillbefedandhowoftentheywillbefed.Horseswillbehousedinpairsinpurposefittedgrasspaddocksapproximately30metersby6metres

Theywillhaveaccesstograsssupplementedwithlucernehay2.5%bodyweightfedtwicedailyandhavefreeaccessotwater.72-Wherewillproceduresbeperformed?

Inapurposebuiltlargeanimalsurgicalcomplex.Thewoundswillbedressedandphotographedinthetreatmentareaofthehospitalcourtyard.

73-Willanimalsbeheldinaprocedureroomatanypointduringthisprotocol?No

74-Pleaseprovidedetailsofenvironmentalenrichmentsuppliedtoanimalsduringtheprotocol.Ifyouarenotusingenvironmentalenrichment,pleaseprovidereasons.Horsesareacclimatedandrunasaherdandkeptinpairs.Evenwhenkeptaspairstheywillalwaysbeinsightoftheotherhorses.77 - Have any researchers named on this application had their AEC approvalwithdrawn?

No78-PleasespecifytheroleoftheChiefInvestigatorinthisprotocol(e.g.supervisiononly,anaesthesia/surgicaltechniquesetc)DrDartwillperformthesurgeryandispresentandassistinginallprocedures.DrDartiscontactable24/7andisthefirstpointofcontactforanyissues80-PleaseprovidedetailonthelevelofexperienceoftheChiefInvestigatorwithtechniques to be performed on live animals in this protocol.For each techniquepleasespecifythelevelofexperience,stepstobetakentoincreaseexperience,andthe name of senior staff on the protocol who will supervise for unfamiliarprocedures.

Dr Dart has been an equine veterinarian for 32 years, a large animal surgicalspecialistfor23yearsandaclinicianandresearcherforhiswholecareer.Heisanexpertatalltheprocedures.

Heisthepointofcontactforanydecision

81-Forallinternalcoinvestigators,pleaseprovidedetailonthelevelofexperiencewithtechniquestobeperformedonliveanimalsinthisprotocol.Foreachtechniqueplease specify the level of experience of the investigator, steps to be taken toincreaseexperience,andthenameofseniorstaffontheprotocolwhowillsuperviseforunfamiliarprocedures.

DrTsanghasbeenaveterinarian for5yearsandworkedwithhorsesanddoingresearch for that time. He is training as a specialist and is competent in all

proceduresperformed.82-Forallexternalcoinvestigators,pleaseprovidedetailonthelevelofexperiencewithtechniquestobeperformedonliveanimalsinthisprotocol.Foreachtechniqueplease specify the level of experience of the investigator, steps to be taken toincreaseexperience,andthenameofseniorstaffontheprotocolwhowillsuperviseforunfamiliarprocedures.

NA84-ExperimentalProtocol(non-wildlife)Wherewilltheanimalsbeobtainedfrom?

Saleyard86-Pleasespecifywhereanimalwillbeobtainedfrom(includingprotocolnumberifapplicable)

Thehorseswillbesourcedfromsaleyards87 -Will the protocol involve the use of geneticallymodified (gene knockout ortransgenic)animalsorgeneticallymodifiedmicro-organisms?

No88 - <div align="left">Thank you for completing the University of Sydneyâ€™sAnimalEthicsApplicationQuestionnaire.Afterselecting'Completed'below,pleaseremember to attach anydocuments relevant to your application in the next tab.After completing your application, return to the 'Coversheet' tab and press the'Submit' button. You will receive an email shortly after confirming yoursubmission.Once again, if you require further information on the applicationprocedure, please consult our <ahref="http://sydney.edu.au/research_support/ethics/animal/">website</a> oremailtheAnimalEthicsteamatanimal.ethics@sydney.edu.auIfyouexperienceanytechnicaldifficulties,pleasedonothesitatetocontactResearchSupportusingthedetailsbelow:T+61286278183Eresearch.support@sydney.edu.au</div>

Completed

ATTACHEDDOCUMENTS

DateUploaded Type DocumentName11/10/2016 OtherHorseRecord

11/10/2016 OtherHorsewouldmeasurements

11/10/2016MonitoringSheet MonitoringSheet

Appendix2:Anaestheticmonitoring

Infusions

COMMENTS CODES

HR •

RR SPONT O,

RR IPPV ∅,

Blood pressure:

Systolic V,

Diastolic ^,

Mean –,

Sp02 (%)ETC02 (mm Hg)TEMP (°C)

FLUIDS ml/hr

ET ANAE (%)

PRE- OXYGENATE !ET Size: mm Cu"ed ! BREATHING SYSTEM+ FGF:

! RBS ................ L/min

! NRBS ............. L/min

BODY POSITION:

! D ! LL ! RL ! S

MONITORING:

! PULSE OX

! ETC02

! DOPPLER

! ET ANAES

! TEMP

ANALGESIA

BP CUFF SIZE:

TEMP SUPPORT:

! AIRBED

! HEAT PAD

! WARM AIR BLANKET

! OTHER .....................

COMMENTS

IV FLUIDS

TYPE: ............................

Tot mls: .........................

TIME TO:

Start Proc. 1 ..................

Start Proc. 2 .................

End Surgery ..................

COMPLICATIONS:

! Hypotension

! Hypoventilation

! Hypoxaemia

! Hypothermia

! Bradycardia

! Arrhythmia

! Blood Loss

! Other

SA RECOVERY:Time In Recovery .........................................

Temp ..............................

Extubation Time.........................................

Recovery Quality:! Good

! Poor

! Satisfactory

PREMEDICATIONDrug Dose - mg/kg = Total mg = Total ml Route Time

SEDATIONQUALITY! Profound! Moderate! Poor

INDUCTIONDrug Total ml admln = Total mg = mg/kg Route Time

TIME :00 :15 :30 :45 :00 :15 :30 :45 :00

TEMP °C HR/PULSE RATE MM/CRT RR PCV PROTEIN USG

UREA CREAT PHYSICAL STATUS1 2 3 4 5 E

WEIGHT ABNORMAL LAB WORK

COMMENTS

THE UNIVERSITY OF SYDNEY VETERINARYTEACHING HOSPITAL CAMDEN

ANAESTHESIA RECORD

EQUINE RECOVERY:

Time In Recovery ....................

Extubation Time ......................

Time Sternal ............................

Drugs Administered ................ ...................................................

Time Standing .........................

Recovery Quality: A— B— C— D— E— F— G— H— Total:—

Appendix3:Horserecords

HORSERECORD

TagNumber:Weight:DateofTetanusprophylaxis:DateofAnthelminticadministration:DateofSurgery:

WOUNDTEMPLATEWound LeftLeg RightLegWound1 Wound2 Abbreviations:UMF20–manukahoneyUMF20CBD–CannabidiolTHC-TetrahydrocannabinolCO–control(saline)

Appendix4:Post-operativemonitoring

HORSEMONITORINGSHEETHorsetagnumber:Date Attitude Gait Swelling Comments Anyhorsesthatarenotbrightandalertwillbegivenafullclinicalexaminationandtreatedappropriately.Anyhorseswithlamenesswillbegivenafulllamenessexaminationandtreatedappropriately.Ifthereisexcessiveswellingandlamenesssuggestiveofcellulitishorseswillbegivenafullexaminationandtreatedappropriately.

HorseWoundMeasurementsHorseTagNumber

WoundSize(cm2)

LFUpper

LFLower RFUpper RFLower

Week1Day1

Totaldaystohealing

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