Dunham, J. , Sales, A., & Pickering, A. E. (2018

Dunham, J., Sales, A., & Pickering, A. E. (2018). Ultrasound-guided,open-source microneurography: Approaches to improve recordingsfrom peripheral nerves in man. Clinical Neurophysiology, 129(11),2475-2481. https://doi.org/10.1016/j.clinph.2018.07.011

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https://doi.org/10.1016/j.clinph.2018.07.011

https://doi.org/10.1016/j.clinph.2018.07.011

https://research-information.bris.ac.uk/en/publications/1a8feefd-887c-435b-bd61-182b1499a32e

https://research-information.bris.ac.uk/en/publications/1a8feefd-887c-435b-bd61-182b1499a32e

Dunhametal.Ultrasoundguidanceinmicroneurography. 1

Ultrasound-guided,open-sourcemicroneurography:Approachestoimproverecordingsfromperipheralnervesinman.JamesP.Dunhama,b*,AnnaC.SalesaandAnthonyE.Pickeringa,b.a-SchoolofPhysiology,Pharmacology&Neuroscience,UniversityofBristol,Bristol,UnitedKingdom.b-Anaesthesia,Pain&CriticalCareSciences,TranslationalHealthSciences,BristolMedicalSchool,UniversityofBristol,BS28HW.Correspondingauthor(*)[email protected]:ThisworkwassupportedbytheNationalInstituteforHealthResearchandWellcomeTrust[gr088373].JPDisanNIHRAcademicClinicalFellow.


StructuredabstractObjectiveMicroneurographyistheonlymethodforrecordingfromsingleneuronsinintacthumannerves.Itischallenging-requiringtechnicalexpertise,investmentinspecialisedequipmentandhassparsedatayields.MethodsWeassessedwhetherultrasoundguidanceincombinationwithan‘openaccess’amplifieranddatacapturesystem(Open-Ephys)wouldsimplifyandexpandthescopeofmicroneurographicrecordingsinhumans.ResultsIn32healthyconsentingvolunteers,ultrasound-guidanceimprovedsuccessratesforobtainingcutaneousC-fibresandreduced“SkintoNerve”timesfrom28.5minutesto4.5minutesforrecordingsoftheperonealnerve(P<0.0001).Weillustratethepotentialutilityofultrasound-guidedmicroneurographyfordifficulttoaccessnerveswithphrenicnerverecordingduringaValsalvamanoeuvre.WeshowthatOpenEphysisaviablealternativetocommerciallyavailablerecordingsystemsandoffersadvantagesintermsofcostandsoftwarecustomisability.ConclusionsUltrasoundguidanceformicroneurographywithOpenEphysfacilitatescutaneousCnociceptorrecordingsandallowsrecordingstobemadefromnervespreviouslyconsideredinaccessible.SignificanceWeanticipatethattheadoptionofthesetechniqueswillimprovemicroneurographyexperimentalefficiency,addsanimportantvisuallearningaidandincreasesthegeneralisabilityoftheapproach.Keywords:Microneurography,Pain,C-fibre,Nociceptor,Open-source,Ultrasound.HighlightsUltrasound-guidedmicroneurographyimprovesdatayieldsandreducesexperimentalduration.OpenEphys,anopensourcedataacquisitionsystem,offersadvantagesforhumanmicroneurography.UltrasoundwithOpenEphyswillfacilitatetargetedmicroneurographybaseduponclinicalpresentation.1.IntroductionMicroneurographyisanestablishedtechniquethatallowsrecordingstobemadefromperipheralnervesinhumans(VallboandHagbarth,1968,Vallboetal.,2004).ThisincludestheabilitytorecordfromafferentneuronesconductingintheC-fibrerange(TorebjorkandHallin,1974),whicharepredominantlynociceptors.Inthepainfield,thenormalresponsecharacteristicsofseveralclassesofnociceptorshavebeendefined


andagrowingbodyofworkisnowidentifyingpathophysiologicalchangesinhumandisease(DonadioandLiguori,2015).Despitetheseadvancesandmanydecadesofuse,microneurographyremainschallenging.Inthewordsofthedevelopersofthetechnique,“..experimentsaretimeconsuminganddemandingforboththeexperimenterandthesubject,particularlywhentheaimistostudysingle-unitactivity.Shortrecordingsessionsmaybeprecededbylongsearchperiodsrequiringmaximalattentiontodelicatevisualandauditorycueswhiletheexperimenterhastomakeminutemanualadjustmentsoftheelectrodeposition.”(Vallboetal.,2004).Anadditionaldifficultyistheexpenseandrelativepaucityofchoiceofavailablehardwareandsoftwarewhichiseithercustombuilt/writtenoravailablefromonlyahandfulofsupplierscommercially.Theuseofultrasoundtoaidtheplacementofmicroneurographyelectrodeshasbeenreported(CurryandCharkoudian,2011,Granataetal.,2016).CurryandCharkoudiantargetedthecommonperoneal,ulnar,medianandradialnerves(largenervescommonlytargetedbymicroneurographers)inordertomakerecordingsofmusclesympatheticnerveactivity(MSNA).Promisingly,theyreportedthat“timetoplacingthemicroelectrodeintothenervecanbereducedtojustafewminutes”however,noquantitativedatawerepresentedanditisunclearhowmanyparticipantswereinvolvedintheirstudy.Alettersubsequentlysuggestedthathigherfrequencyultrasoundscanratescanimproveelectrodevisualisationduringmicroneurographybasedonasinglecaseatthemediannerve(Granataetal.,2016).WewishedtousemicroneurographytorecordfromcutaneousCfibrenociceptorsinnervatingtheskinofthelowerlimb.Inseekingtooptimiseourtechniquewecomparedthetraditionaltechniqueofrecordingfromthecommonperonealnerve(CPN-amixednerve)atthefibularhead,withultrasoundguidedelectrodeplacementinthesuperficialperonealnerve(SPN-asmall,cutaneousnerve)targetedinthelowerleg.Inprinciple,theSPNinthelowerlegshouldyieldahigherproportionofcutaneousCfibrerecordingsasitlacksthemotorfasciclesorsensoryinnervationofdeeperstructures.TheseprincipleshavebeenborneoutpreviouslywithtargetingoftheSPNattheankle,see(Serraetal.,2004)forexample.Furthermore,ithasbeensuggestedthatresearchersmaysuboptimallyplacetheirelectrodesinordertofacilitatevisualisationoftheelectrodewhenusingultrasound.Thiscouldthenleadtounstablerecordings(McNultyandHodson-Tole,2016).Toaddressthispossibility,wealsocomparedultrasoundguidedelectrodeinsertionintotheSPNwithultrasoundlocalisationoftheSPNtoassistamoretraditionaliterativeinsertionprocess.Inadditiontooptimisingourmicroneurographytechnique,wealsotestedthefeasibilityofanalternativeamplifier/dataacquisitionsystem.Classically,microneurographicrecordingshavebeenmadewith‘homemade’amplifiersandanaloguetodigitalconverters.Alternatively,commercialacquisitionsystemsareavailablee.g.NeuroAmpEXfromADIinstruments.Thefirstoptionrequiresconsiderabletechnicalexpertiseandsupport.Thesecondoption,asnotedpreviously(Gloveretal.,2017),hasassociatedhighercostsandexperimentalflexibilityispotentiallyconstrainedbyclosed-sourcesoftware;whichisrequiredforcommercialviability.Theseissuesarenotuniqueto


microneurographyandtheneurosciencecommunityhasincreasinglybeguntoadoptopen-sourcehardwareandsoftwaretools.OnesuchprojectisOpenEphys,whohavedevelopedinexpensivehardwareandfreelyavailablesourcecodeforelectrophysiologicaldataacquisitionandanalysis(webpage:http://www.open-ephys.orgorwiki:https://open-ephys.atlassian.net/wiki/spaces/OEW/overview)(Siegleetal.,2017).Thishardware/softwarecombinationhasbeenwidelyadoptedforanimalneurophysiologystudiesandrecentlyprotocolsforitsuseforEEGrecordingsinhumanshavebeenpublished(Blacketal.,2017,Hermizetal.,2016).Toourknowledgethissystemhasnotbeenusedforhumanmicroneurographywhereitmayofferadvantages.Wedemonstratethatultrasoundguidance,combinedwithanopensourcedataacquisitionsystem,formicroneurographyfacilitatesidentificationandrecordingofnervestoincreasedatayield.Thishasthepotentialtobroadentheutilityofautonomicandsensorymicroneurographybyenablingtargetingofdeepernervesorsmallerperipheralnervesasrequiredbythescientifichypothesisundertestorbytheclinicalpresentationofapatient.2.MethodsExperimentswereconductedinaccordancewithTheCodeofEthicsoftheWorldMedicalAssociation(DeclarationofHelsinki)forexperimentsinvolvinghumans.EthicalpermissionswereobtainedfromtheFacultyofBiomedicalSciencesResearchEthicsCommitteeattheUniversityofBristol(ethicsreferencenumber:51882).Writteninformedconsentwasobtainedfromallparticipants.ExperimentswereperformedattheClinicalResearchandImagingCentre,UniversityofBristol,UK.32healthyvolunteers(F:M22:10,agerange19-34yrs)participatedintheseexperiments.Nosubjectsreportedahistoryofneurologicaldisease.Thermalthresholdsforthelowerleg(L5dermatome),assessedwithacontactthermode(MedocTSA-II,Israel),wereallinthenormalrange:colddetection29.2±1.6˚C,warmdetection36.1±2.5˚Candheatpain42.9±3.2˚C(mean±SDfromabaselineof32˚C).Participantswereseatedcomfortablyinarecliningbed.Therightlegwassupportedwithpillowsandblanketssuchthatitwaskeptrelativelystillandaccesstotherecordingsite(s)wereoptimised.Theskinovertherecordingsitewascleanedwithchlorhexidine,theelectrodesweresterilisedbypriorautoclavingandanotouchtechniquewasemployed.Areferenceelectrode(UNA35FRS,FHC,Maine,USA.)wasinsertedsubcutaneouslyneartheplannedrecordingsite.Inexperimentstargetingthecommonperonealnerve(CPN),thenervewasidentifiedbypalpationproximalandlateraltotheheadofthefibula.ThenervewasstimulatedtranscutaneouslyusingamonopolarsteelsearchcathodewithanECGelectrodeplacedonthemedialaspectofthekneeastheanode.Pulsatilecurrentstimulation(0.5msduration,0-10mA,and0.5Hz)wasappliedtotheskinoverlayingthenerveuntildorsiflexionoftheanklewasevoked(usingaDS7,Digitmer,WelwynGardenCity,UKandaPulsePal,opensourcestimulusgenerator,Sanworks,NewYork,USA).Thecurrentwasreducedtoidentifythelocationwheretheleaststimulusevokedaresponse;thispointwasthenmarkedwithapen.Thenervewasmappedinthis


mannerovertwotothreecentimetresproximaltothefibularhead.Therecordingelectrode(200µmdiameter,35mmlength,tungsten,highimpedance–‘NoZap’;UNA35FNSFHC,Maine,USA.)wastheninsertedintothenerve.Successfulneuralrecordingwasdetectedbythecharacteristicinsertiondischargeassociatedwiththeabilitytoevoke‘massactivity’withgentlemechanicalstimuliovertheskinreceptivefield.Thetimefrominsertingtheneedleintotheskintosuccessfulneuralrecordingswasmeasured(SkintoNervetime).Ifmassactivityindicatingsuccessfulnerverecordingwasnotfoundwithin60minutesofsearching,theexperimentwasabandoned.Thesuperficialperonealnerve(SPN)waslocatedusingaToshibaAplio500UltraSoundscannerwithaPLT-1204BTlineartransducer(usedathighfrequency-18MHz(Granataetal.,2016)).Wewereguidedbythesono-anatomyoftheSPNasdescribedpreviously(Canellaetal.,2009,Chin,2013).Useoftheultrasoundremovedtheneedforelectricalsearchstimulation.Aninsertionsiteinthedistalhalfofthelaterallowerlimbwaschosen:beforetheSPNpiercesthecruralfasciawherethenervewasclearlyvisualised(butwasnotpalpable);andfreefrombloodvesselswithintheexpectedneedletrajectory(Figure1).Theultrasoundprobewascoveredwithasteriletransparentdressingandcoatedwithsterileultrasoundgel.Therecordingelectrodewasinsertedeitherwithdirectreal-timeultrasoundguidance(i.e.identifyingtheelectrodesubcutaneouslyandthenadvancingthetipintra-neurallyunderdirectvision-Ultrasound(US)Guided)oraftervisualisationofthenerveandmarkingoftheskinandwithoutfurtheruseofultrasound(Ultrasound(US)Located).TherecordingandstimulatingarrangementisshowninFigure1A.Signalsfromthemicro-electrodeswereamplifiedwithanIntanRHD2216chip(IntanTechnologies,LosAngeles,USA.)placedclosetotherecordingsiteandgroundedviaanearthplatepositionedonthecalfdistaltotherecordingsite.ThesignalwasdigitisedontheINTANchipat30kHzandrelayedtoanOpenEphys(http://www.open-ephys.org)acquisitionboard.ThedatawasdisplayedviatheOpenEphysGUI(http://www.open-ephys.org/gui/)onalaptopcomputer.Thedigitalbandpassfilterwassetat300-4000Hz.Theneurogramwasalsooutputasanaudiosignalviathelaptopspeakers.Duringdataacquisition,thelaptopwaspoweredfromitsinternalbatteryandtheacquisitionboardwaspoweredviaaUSBPowerbankbattery(ratingof20,000mAhrs,Aukey,https://www.aukey.com).Whensuccessfulneuralrecordingswereobtained,thecutaneousinnervationterritorywassearchedforslowlyconductingafferentsusingtranscutaneousstimulationwithasteelelectrodeofeither1mmor5mmdiametertip.Thissearchoftenstartedinaregionidentifiedbythesubjectasbeingthesiteoforiginofthetransientparaesthesiaelicitedbytheelectrodeadvancement.Thelatencytospikedischargefollowingelectricalstimulationofthereceptivefieldwasnotedandthislatencywasdividedbythedistancebetweenstimulationandrecordingelectrodestogenerateconductionvelocities.Whenslowlyconductingafferentshadbeenidentified(conductionvelocities<1.5m/s),sterileelectro-acupunctureneedles(HarmonyMedicalClassicOriginalAcupunctureNeedles13mmLength-0.22mmDiameter)wereinsertedintradermally,immediatelydistalandadvancedtowithin5mmoftheelectricalreceptivefield.Cfibreswerestimulated(0.125-2Hz,0.5msduration,0-20mA(DigitimerDS7drivenbyPulsePal))atincreasingfrequencytodeterminetheextentoftheiractivitydependentslowing(ADS).The


protocoltoelicitADSwasasperObrejaetal.,2010(Obrejaetal.,2010).ThePulsePalwascontrolledviaMatlab(2016a,Mathworks);stimulationcurrentoutputwasrecordedbyOpenEphysinparallelwithneuralrecordings.Thestimulationprotocolconsistedofa2minutepauseinstimulationfollowedby20consecutivestimulationseachat0.125Hz,0.25Hz,and0.5Hz.Thiswasthenfollowedbyafurther2minutepauseandtheneither2or3minutesofstimulationat2Hzwith60stimulationsat0.25Hz.SensoryafferentdatawereanalysedinMatlab.Spikethresholdsweredeterminedmanuallytoexcludenoiseandwereapproximately+/-10μV.Allthresholdcrossingsfollowingstimulationwerethenplottedastheirlatencyafterthestimulusagainstthetimeofthestimulus.Characteristicconstantlatencythresholdcrossings,whichslowedwithincreasingstimulusfrequency,couldthenberecognisedaslikelyCnociceptoractionpotentials(Geeetal.,1996,Serraetal.,1999)ThesewerethenisolatedfromthenoiseusingtheMatlabfunctionSelectdata.Isolationofthesecharacteristiclatencyresponsesthenallowedforisolationoftheindividualactionpotentialprofiles.Thesewereoverlaidandaveragewaveformsgenerated.Constantlatencyresponseswerealsovisualisedvia“waterfallplots”(TorebjorkandHallin,1974)wheresequentialrawdatatracesfollowingcutaneousstimulationareplotted.ExampleMatlabscriptsareavailablefromthecorrespondingauthoronrequest.Totestwhetherultrasoundguidancecouldconferanadvantagewhenattemptingtoaccessachallengingsiteformicroneurography,wemaderecordingsfromtherightphrenicnerveintheneck(AEP–lastauthor(age53yrs,male)).Thephreniccanbevisualiseddeeptothesternocleidomastoidandsuperficialtotheanteriorscalenemuscle.Itrunslateraltomedialastheprobeismovedcaudally.Itisoftenvisualisedduringinterscalenebrachialplexusblocksforregionalanaesthesiaoftheupperlimb.Indeed,itisofteninadvertentlyblockedbythistechnique(https://www.nysora.com/ultrasound-guided-interscalene-brachial-plexus-block-2017).Theparticipantlaysupinewiththeirheadturnedtotheleft.TheAplioultrasoundscannerwasusedtovisualisethenerveatasite2cmrostraltotheclavicle.Atthispoint:theprobecouldbeheldcomfortablywithgoodqualityimages;therewaslargeseparationbetweenthephrenicnerveandthebrachialplexusandtherewerenooverlyingbloodvessels.Theinsertionsitewascleanedwith2%chlorhexidine.Thereferenceelectrode(UNA35FRS–asforthelowerlimbnerves)wasinsertedoverthemastoidprocesstominimiseEMGinterference.Therecordingelectrode(UNA35FNS)wasinsertedlateraltomedialandin-plane.Theinsertionoftheelectrodetothenervewasuneventfulandcausedminimaldiscomfort.Withtherecordingelectrodestablyplacedwithinthenerve,thesubjectperformedanumberofrespiratorymanoeuvresincludingincreasinglylargeinspiratoryefforts,Valsalvaandheldinspiration.ECGwassimultaneouslyrecorded(leadII)viaself-adhesiveECGelectrodesfixedtoeachshoulderandthesignalwaspassedtotheIntanChip.ThesedatawereconvertedintoabinaryfilewithinMatlab(scriptavailableonrequest)andthenimportedintoSpike2v7(CambridgeElectronicDesign).Therawnervesignalwasdigitallybandpassfiltered(300Hzto4kHz)andtheECGat(0.1–25Hz).Spikewas


thenusedtodisplaythedataandtoextractinstantaneousphrenicfiringrates(0.2secondrollingaverage)andinstantaneousheartrate.Alldatawereexpressedasmean±SEMunlessotherwisestated.TimestosuccessfulnerverecordingsweredisplayedasKaplanMeierplotsandanalysedusingtheMantel-Coxtesttoaccountforcensoredcases(wherenorecordingwasobtainedwithinthepre-definedcutoffperiod(ChoiandLam,2017)).TheseanalysesandcomparisonsofpainratingswereperformedinPrism5(GraphPad,SanDiego,USA).


3.ResultsandDiscussionTheexperimentalinterventionsrequiredtomakethemicroneurographicrecordingsweregenerallywelltoleratedbythestudyparticipantsandwerecompletedin88%ofsubjects(28/32).Fourrecordingswereterminatedearly:onevolunteerfeltlightheadedfollowingneedleinsertionintothenerve,onecomplainedofpainonneedleinsertionandtwowereunabletostaysufficientlystilltocompletetheexperimentalprotocol.Atfollowup,oneweekafterrecordings,noneofthesubjectsreportedanypersistingpain,discomfort,numbnessorparaesthesia.DuringourinitialeffortstomakecutaneousCnociceptorrecordings,wefoundthatrecordingsfromthecommonperonealnerveoftenyieldednon-cutaneousafferents,i.e.fromfibresbelongingtothedeepperonealnerve(DPN).Intotal63%(10/16)ofrecordingsfromtheCPNyieldedDPNafferents.Wewerethereforeinasituationwherewewerefindingnerverecordingstimeconsumingtomakeand,whenwecouldmakerecordings,theidentifiedunitwasnotofthecutaneoussubtypethatwewereseeking.ToimproveourdatayieldswewishedtotargettheSPNdirectly,however,itssmallsizemakesitchallengingtorecordusingconventionalpalpationandelectricalstimulationmethods(ithasnomusclefasciclessonotwitchisevokedalthoughevokedparaesthesiacouldbeusedtoidentifyelectrodeproximity).Thesono-anatomyoftheSPNiswellcharacterised(Canellaetal.,2009,Chin,2013)anditiscommonlytargetedduringregionalanaesthesiaaspartofanankleblocksoweswitchedtoattemptingtouseultrasoundtolocatethenerve.TheSPNwasstraightforwardtolocateonultrasoundscan(Figure1,BandC).Thetimetoidentifythenervewithultrasoundwasnotspecificallyrecorded,butwefoundthistookonlyacoupleofminutesmaximum.HoldingtheSPNintheshortaxisviewallowedtherecordingelectrodetobeadvanced‘inplane’fromtheanterioraspectofthelowerlegaimingposteriorlytowardsthenerve.Thisapproachenabledvisualisationoftherecordingelectrodealonganapproachtrackofupto1cmoftissuetostabilisetheelectrodeafterinsertionintothenerve(Figure1).Itisworthnotingthattheuseofultrasoundremovedtheneedtoelectricallystimulatethenervewhilsttryingtolocateitsposition.Thisbothshortenstheexperimentdurationandreducestheunpleasantnessandstressfortheparticipant.Ultrasoundguidancesignificantlyincreasedthenumberofexperimentsresultinginsuccessfulnerverecordings.Furthermoreitsignificantlyreducedthetimetakentoachievesuccessfulrecordings.Successratesandmedianskintonervetimesimprovedfrom80%and28.5minutesattheCPN(18participants)to100%and4.5minutesattheSPN(6participants)(P<0.0001,Mantel-Coxlogranktest,Figure2A).Additionally,incomparisontousingtheultrasoundtoonlylocate(Ultrasound(US)located)theSPN,fullreal-timeultrasoundguidance(Ultrasound(US)guided)improvedsuccessratesfrom20%(1outof5,withthetimetakeninthatonesuccessof9mins)to100%withamedianskintonervetimeof7(range2-9)minutes(P=0.005,Mantel-Coxlogranktest,Figure2B).Thereforetheuseofultrasoundguidancewasbeneficialbothintermsoftimeandyieldofrecordingsfromtheperonealnerveirrespectiveofthechosencomparatorsite.NodifferenceswerefoundinthepainscoresfromthevolunteerswhoparticipatedintheexperimentstocompareultrasoundguidancetoultrasoundlocationoftheSPN:


Worstpain(0-10)ultrasoundguidedvsultrasoundlocated3.4±1.2vs3.4±1.2andaveragepain(0-10)1.5±0.5vs1.4±0.2respectively(n=5).Oneconcernthathasbeenexpressedabouttheuseofultrasoundinmicroneurographywasthatitcouldnotguaranteeimproveddatayields,onlyincreasetheefficiencyofplacingtherecordingelectrodeintothenerve.Itcouldalsotheoreticallyreducethestabilityoftherecordingsiftherecordingelectrodetrajectorywasalteredtomakeinsertionamenabletoultrasoundguidance(McNultyandHodson-Tole,2016).CutaneousCfibrenociceptorswereidentifiedin9/13experimentswithultrasoundguidancewithintheSPNand2/18withtheclassicapproachattheCPN.OftenthereweremultipleconstantC-fibrelatencieswithintheserecordings;presumablytheelectrodetipwasinthevicinityofaRemakbundle.Thoughmostoftenonlyoneunitwasidentifiedwiththetranscutaneousstimulationanditwasthisunitthatwasthesubjectoffurthercharacterisation.Thechosenunitwasselectedbasedonanamplitudethatwassufficientlylargetobeidentifiablebyeyeduringstimulation,i.e.asignal:noiseratioofgreaterthan~1.5.Wedidnotnoteanyproblemswithstability.Weheldtheseunitsfor65(50–85)minutes(mean(range))bothattheCPNandtheSPNuntiltheendofourstudyprotocol.Onlyinoneoftheserecordingswastheunitlostpriortocompletionoftheprotocol.Astheexperimentswereterminatedattheendoftheprotocol,theupperrangeofourrecordingtimesdoesnotrepresentmaximalpossiblerecordingtimes.Importantly,wedidnotloseanyrecordingswhenremovingtheultrasoundprobefromcontactwiththelegwhichmightbeexpectedtobethecriticalpoint.AnexampleofaCfibrenociceptorrecordingfromtheSPNusingtheOpenEphysacquisitionsystemisshowninFigure3.Theshortestpossibleconductiondistancewas0.18mgivinganestimatedinitialconductionvelocityof0.9m/s.ThedegreeofslowingfrombaselineintheCfibreduringbothlowfrequency(0.6%at0.125Hz,1.2%at0.25Hzand2.4%at0.5Hz)andhighfrequency(~23%at2Hz)stimulationsuggeststhatthisunitwasnociceptive(Obrejaetal.,2010).Thesignaltonoiseratiowassufficientlygoodtoisolatethespikewaveformfromnoisewithasimplethresholdcrossingcriteriontobuildtheactivity-dependentslowing(ADS)profile(figure3A).IndividualCfibreADSprofilescouldthenbeselectedfromtherasterplotandisolatedforfurtheranalysis(Figure3B);forexample,averagewaveformsfromtheADSperiodcouldbegenerated(Figure3D).A‘waterfall’plotofrawdatatracesfromsequentialstimulationsduring2Hzstimulationisalsoshown(Figure3C).Figures3Cand3DdemonstratethesignaltonoiseratiosusingtheOpenEphys/Intanacquisitionpackage.Thebaselinenoiseinthistrace,oflessthan±10µV,ischaracteristicoftheOpenEphysacquisitioninourhands.Itisofnotethattheseexperimentswerenotperformedinanelectricallyshieldedlab.ThecompleteOpenEphysacquisitionsystemwithIntanamplifierchipandthePulsePalstimulusgenerator,costapproximately£4000.Thiscompareswellwithequivalentlyspecifiedcommerciallyavailablesystems.Itofferspotentialperformanceadvantagesbecauseoftheonchipamplificationanddigitisationneartherecordingsitewhichreducesnoise/hum.Inaddition,theopensourcestructureofthesoftwareprovidesanopportunityforcustomisationofalgorithmsforacquisitionandanalysis.


Theutilityofultrasoundguidedmicroneurographyinaccessingdifficulttoreachnervesisdemonstratedbytherecordingsmadefromthephrenicnerve.Tothebestofourknowledgethisisthefirstreportofsucharecording.ThegrossandultrasoundanatomyoftheapproachisshowninFigure4AandB.Themulti-unitphrenicnerveactivity(Figure4C)showsarampingpatternwithamplitudegradedaccordingtoincreasingrespiratoryeffortwhichismaximalduringamodifiedValsalvamanoeuvre(withaccompanyingheartratechanges).Therecordingwasstablyandcomfortablyheldduringthesemanoeuvresforaperiodofalmostanhour.Priortoundertakingourrecordingsfromthephrenicnerveweperformedariskassessment.Microneurographyhasanexcellentsafetyrecordwithnolongtermmotororsensoryabnormalitiesreportedwhenconductedbytrainedspecialistswithappropriatecareandconsideration.Wedoknowthatmicroneurographycarries<10%riskofparaesthesialasting2-10days(Eckbergetal.,1989,GandeviaandHales,1997,Vallbo,2018).Whenappliedtothephrenicnerve,weassessedthattheworstpossibleoutcomeofmicroneuorographycouldbeahemi-diaphragmpalsysuchasthosereported(relativelycommonly)withbrachialplexusregionalanaesthesia(El-Boghdadlyetal.,2017).Otheranticipatedadverseoutcomesincludedhiccoughsandpain.Aresolvinghemi-diaphragmpalsyisonlyofclinicalrelevanceinpatientswithunderlyingrespiratorydiseaseas,“Inhealthyindividuals,…..tidalvolumesremainunchangedduetoagreatercontributionfromtheribcage”(El-Boghdadlyetal.,2017).Therefore,althoughphrenicpalsywasourworstcasescenario,weanticipatedthatitwasveryunlikely(giventhelowincidenceofsuchpalsieswithmicroneurographyatothersites)andthatevenshoulditoccurthenitwouldbeunlikelytocauserespiratorycompromiseinanotherwisehealthyvolunteersubject.Tofurthermitigaterisk,ourexperimentswereperformedinaclinicalresearchinstitutethathasfullresuscitationfacilities.Inthiscasetherecordingproceededuneventfully,waseasilytoleratedandproducednoshortorlongtermsequalae.Nonethelesswerecommendthatanyeffortstorepeatsuchphrenicrecordingsproceedonlyinthecontextofappropriateriskassessmentandmitigation.Asdemonstratedhere,ultrasoundguidance,combinedwithopensourcedataacquisitionsystemshasthepotentialtosimplifytheadoptionandbroadenthescopeofautonomicandsensorymicroneurography;enablingtargetingofdeepernervesorsmallerperipheralnervesasrequiredbythescientifichypothesisundertestorbytheclinicalpresentationofapatient.


4.ConclusionsandSignificanceWehavedemonstratedquantitativelythatultrasoundguidedelectrodeinsertioninsensorymicroneurographyimprovesbothdatayieldsandproductiveexperimentaltime.Itshouldbenotedthatwehaveexaminedthebenefitsofultrasoundinthehandsofarelativelyinexperiencedmicroneurographer(JPD,5monthsprevioustraininginanestablishedmicroneurographylab)andinthecontextofestablishingthetechniqueofsensorymicroneurographyfromscratchinourlaboratory.Basedonourexperienceweanticipatethatnovicemicroneurographersmayfindbenefitinusingultrasoundtolocatetheirtargetnervesinthatitmayshortentheirlearningcurveandgivethemmorerealtimefeedbackinformationonwhichtodeveloptheirpractice.WehavealsoprovidedevidencethattheOpenEphyselectrophysiologicaldataacquisitionsystemissuitableformicroneurographyandenableslownoise,stablerecordings.Therecordingsystemiscosteffectivecomparedtocommerciallyavailablemicroneurographysystems,isrelativelyportableand,asthesoftwareisopensourceitcanbemodifiedtomeettheneedsoftheresearchcommunity.Inasingleindividual,wehaveperformedaproofofprinciplestudywhichdemonstratestheutilityofultrasoundguidanceintargetingthephrenicnerve,whichhaspreviouslybeenconsideredinaccessibletomicroneurography.Thisapproachwaswelltoleratedanddemonstratedtheclassicalphysiologyasexpectedfromprioranimalwork.Infuturestudieswehopethatultrasoundguidancewillnotonlyimproveexperimentefficiency,butwillalsofacilitatetargetingofpreviously‘challenging’nervesasdictatedbythespecificresearchquestionorbyclinicalpresentation:forexamplesmallcutaneousnerveswithdistalneuromasordeepmuscleinnervation.Assuchevenexperiencedmicroneurographersmayfinditofusetoaccessnewtargetnervesandfacilitatepatientstudies.5.AcknowledgementsDr.KennethK.F.Ho:ONLINEMRI&CTSECTIONALANATOMY,http://omcsa.org.FortheMRIimagesincludedinfigure1.Noneoftheauthorshavepotentialconflictsofinteresttobedisclosed.


Figure1-Ultrasoundguidedplacementofthemicroneurographyrecordingelectrodeinthesuperficialperonealnerve.A.Diagrammaticrepresentationoftheexperiment.Thehighfrequencylinearultrasoundprobeisplacedlateraltotheshinoverthesuperficialperonealnerve(SPN)withitslongaxisorientedanterior-posteriorallowingvisualisationoftherecordingelectrodeinplane(seepanelB).ThesignalfromtheactiveandreferenceelectrodesareamplifiedanddigitisedbytheIntanRHD2216chip,positionedclosetotherecordingsite.ThedigitisedsignalisrelayedviatheOpenEphysacquisitionboxbeforebeingdisplayedandstoredonabatterypoweredlaptop.ThelaptopcomputeralsorunsMatlab2016bwhichdrivesthePulsePalstimulusgeneratortriggeringtheDS7constantcurrentstimulator.ThisstimulationisdeliveredtothecutaneousreceptivefieldoftheCfibreonthedorsumofthefootviafineintradermalneedles.B.Imagesobtainedduringultrasoundguidedneedleinsertion(structuresannotatedbelow).Theelectrodecanbeseeninthelongaxisenteringsuperficiallyandanteriorlyinthetopleftofthepaneltowardsthehyper-echoicSPN(~5mmdiameterwithvisiblenervefascicles).TheSPNisflankedanteriorlybyextensordigitorumlongus(EDL)andposteriorlybytheperoneusmuscles(PM).ThefibulacanbeseendeeptoPMinthebottomrightofthepanel.C.CrosssectionalMRimageofthelowerlegforreferenceshowingtherelativepositionsofthelateralstructuresvisualisedwiththeultrasound.NotethesuperficiallocationoftheSPN.


Figure2–Ultrasoundguidanceinmicroneurographyimprovesdatayieldsandreducestimetosuccessfulrecordings.A.KaplanMeierplotcomparingtimetosuccessfulrecordingswithaclassicapproachtotheCommonPeronealNerve(usingsurfacelandmarks,palpationandelectricalstimulationtolocalise)toanultrasound-guidedapproachtotheSuperficialPeronealNerve.Theclassicapproachwasusedin18volunteersandtheultrasound-guidedin6.Mediantimestosuccessfulrecordingswere4.5minsforultrasoundguidedvs28.5minutesfortheclassicapproach(P<0.0001Mantel-Cox,Log-rankTest).Ultrasoundguidanceledtoa100%successratevs80%withtheclassicapproach(withina1hourpermissiblesearchwindow).B.Insertionoftherecordingelectrodewithreal-timeultrasoundguidanceimprovesyield.In5volunteers,thetimetosuccessfulnerverecordingswerecomparedwhenusingdirectvisiontoguideplacementoftherecordingelectrode(ultrasound(US)Guided)vsusingultrasoundtosimplylocatethenerve,markingtheskinandthenplacingtheelectrodeiteratively(ultrasound(US)Located).Ultrasoundguidanceledtoa100%successratevs20%withtheiterativeapproach.Thesesurvivalcurvesaresignificantlydifferent(P<0.005Mantel-Cox,Log-rankTest).Themediantimetosuccessfulrecordingwas7minsforultrasoundguided.


Figure3–Exampleneuronalrecordings.A.Exampleofactivitydependentslowing(ADS)inaCfibreafferent.Theconductiondistancewasatleast0.18m(conductionvelocityof~0.92m/s).TheleftYaxisshowslatencytothresholdcrossingsfollowingcutaneouselectricalstimulation(blackdots).TherightYaxisshowsthefrequencyofstimulation(reddots).ADSisevokedintheCfibrewithaninitiallatencyof~0.19swithlowfrequency(0.125,0.25and0.5Hz)andthentoagreaterextentwith2HzstimulationRecoveryfromADSisdemonstratedduringsubsequent0.25Hzstimulation.Notethatasecondmoreslowlyconductingafferentisalsoseentobeslowingduring2Hzstimulationatalatencyof0.26to0.28s.B.FollowingextractionusingMatlab,theADSinthenowisolatedCfibreismoreclearlyshown.Theunitslows0.6%at0.125Hz,1.2%at0.25Hz,2.4%at0.5Hzand23%at2Hz.TheverticalgreyrectangleshowsthosesweepsdisplayedinpanelCC.Sequentialrawdatasweepsfrom0.18safterthestimulationto0.3safterthestimulation(xaxis)areshown.Theearliestsweepisuppermost–labelled1.TheYaxisisvoltagewithaminimamaximaofeachsweepof-5µVto+20µV.SlowingoftheCfibreactionpotentialwith2Hzstimulationiseasilyrecognised(solidbox).Furthermore,2additionallikelyCfibreactionpotentialsarealsoseen(dottedboxes).D.AlloftheactionpotentialsidentifiedfromthesingleCfibrenociceptorareoverlaid.Theactionpotentialsarecentredupontheirmaximaldeflectionwhichisshownat1ms.Themeanvoltageisshownasasolidblacklineboundedbythestandarddeviationshownasthepairedblackdashedline.NotethattheCfibresthatwehaveidentifiedhavetheirmajordeflectioninthenegativedirectionasisexpectedfrommicroneurographicalrecordings–thescaleinfigures3Cand3Disinvertedtoshowthemorestandardupwarddeflection.


Figure4-Ultrasoundguidedphrenicnerverecordings.A.Schematicofadissectionoftherightsideoftheneckshowingtherelationsofthemajorbloodvessels(internaljugularvein–I.J.V.andcommoncarotidartery),theanteriorscalenemuscle(A.S.)andthephrenicnervepassingfromrostralandlateraltocaudalandmedialovertheA.S.Theblackrectanglerepresentstheapproximatepositionoftheultrasoundprobe.B.Ultrasoundimageduringelectrodeplacementintothephrenicnerve.TheorientationoftheimageisasshowninA.Thephrenicnervecanbeseenintransversesectioninthecentreoftheimage,highlightedwithayellowcircle.Thediameterisoftheorderof1mm.ItliessuperiorandmedialtotheASandinferiortothesternocleidomastoidmuscle(SCM).Theelectrodecanbeseenjustenteringthenerve–highlightedinthedashedredrectangle.C.Phrenicnerverecording.Multi-unitphrenicnerveactivity(withaveragespikerate)andECGwithheartrateduringperiodsoftidalventilation(TV),aninspiratoryhold(TVhold),maximalinspiration(MaxI)andaValsalvawithforcedexpirationagainstaclosedglottis.Eachinspiratoryperiodismarkedwithaverticalbar.NotetheaugmentingpatternofphrenicdischargeininspirationandthemaintainedactivityduringaninspiratoryholdandduringtheValsalvamanoeuvreearlyphase.




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6.BibliographyBlackC,VoigtsJ,AgrawalU,LadowM,SantoyoJ,MooreC,etal.OpenEphyselectroencephalography(OpenEphys+EEG):amodular,low-cost,open-sourcesolutiontohumanneuralrecording.Journalofneuralengineering.2017;14:035002.CanellaC,DemondionX,GuillinR,BoutryN,PeltierJ,CottenA.Anatomicstudyofthesuperficialperonealnerveusingsonography.Americanjournalofroentgenology.2009;193:174-9.ChinKJ.Ultrasoundvisualizationofthesuperficialperonealnerveinthemid-calf.Anesthesiology.2013;118:956-65.ChoiSW,LamDMH.Comparingtimesinclinicalstudieswithafiniteending.Anaesthesia.2017;72:1554-6.CurryTB,CharkoudianN.Theuseofreal-timeultrasoundinmicroneurography.Autonomicneuroscience:basic&clinical.2011;162:89-93.DonadioV,LiguoriR.Microneurographicrecordingfromunmyelinatednervefibersinneurologicaldisorders:anupdate.Clinicalneurophysiology:officialjournaloftheInternationalFederationofClinicalNeurophysiology.2015;126:437-45.EckbergDL,WallinBG,FagiusJ,LundbergL,TorebjorkHE.Prospectivestudyofsymptomsafterhumanmicroneurography.ActaPhysiolScand.1989;137:567-9.El-BoghdadlyK,ChinKJ,ChanVWS.PhrenicNervePalsyandRegionalAnesthesiaforShoulderSurgery:Anatomical,Physiologic,andClinicalConsiderations.Anesthesiology.2017;127:173-91.GandeviaSC,HalesJP.Themethodologyandscopeofhumanmicroneurography.JNeurosciMethods.1997;74:123-36.GeeMD,LynnB,CotsellB.Activity-dependentslowingofconductionvelocityprovidesamethodforidentifyingdifferentfunctionalclassesofc-fibreintheratsaphenousnerve.Neuroscience.1996;73:667-75.GloverPM,WatkinsRH,O'NeillGC,AckerleyR,Sanchez-PanchueloR,McGloneF,etal.Anintra-neuralmicrostimulationsystemforultra-highfieldmagneticresonanceimagingandmagnetoencephalography.JNeurosciMethods.2017;290:69-78.GranataG,GiambattistelliF,PaduaL,CoraciD,PetriniFM.High-frequencyultrasoundinguidingneedleinsertionformicroneurography.Clinicalneurophysiology:officialjournaloftheInternationalFederationofClinicalNeurophysiology.2016;127:970-1.HermizJ,RogersN,KaestnerE,GanjiM,ClearyD,SniderJ,etal.Acliniccompatible,opensourceelectrophysiologysystem.Conferenceproceedings:AnnualInternationalConferenceoftheIEEEEngineeringinMedicineandBiologySocietyIEEEEngineeringinMedicineandBiologySocietyAnnualConference.2016;2016:4511-4.McNultyPA,Hodson-ToleEF.Letterinresponseto"High-frequencyultrasoundinguidingneedleinsertionformicroneurography"byGranataandcolleagues.Clinicalneurophysiology:officialjournaloftheInternationalFederationofClinicalNeurophysiology.2016;127:1737-8.ObrejaO,RingkampM,NamerB,ForschE,KluschA,RukwiedR,etal.Patternsofactivity-dependentconductionvelocitychangesdifferentiateclassesofunmyelinatedmechano-insensitiveafferentsincludingcoldnociceptors,inpigandinhuman.Pain.2010;148:59-69.SerraJ,CamperoM,BostockH,OchoaJ.TwoTypesofCNociceptorsinHumanSkinandTheirBehaviorinAreasofCapsaicin-InducedSecondaryHyperalgesia.JNeurophysiol.2004;91:2770-81.


SerraJ,CamperoM,OchoaJ,BostockH.Activity-dependentslowingofconductiondifferentiatesfunctionalsubtypesofCfibresinnervatinghumanskin.JPhysiol.1999;515(Pt3):799-811.SiegleJH,LopezAC,PatelYA,AbramovK,OhayonS,VoigtsJ.OpenEphys:anopen-source,plugin-basedplatformformultichannelelectrophysiology.Journalofneuralengineering.2017;14:045003.TorebjorkHE,HallinRG.IdentificationofafferentCunitsinintacthumanskinnerves.Brainresearch.1974;67:387-403.VallboAB.Microneurography-howitstartedandhowitworks.Journalofneurophysiology.2018.VallboAB,HagbarthKE.Activityfromskinmechanoreceptorsrecordedpercutaneouslyinawakehumansubjects.ExpNeurol.1968;21:270-89.VallboAB,HagbarthKE,WallinBG.Microneurography:howthetechniquedevelopedanditsroleintheinvestigationofthesympatheticnervoussystem.JApplPhysiol.2004;96:1262-9.

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Dunham, J. , Sales, A., & Pickering, A. E. (2018