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Platform Development for the Modulation of Epileptic Seizure Base on Interictal Spike Rate

Permanent Link: http://ufdc.ufl.edu/UFE0042553/00001

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Title: Platform Development for the Modulation of Epileptic Seizure Base on Interictal Spike Rate
Physical Description: 1 online resource (103 p.)
Language: english
Creator: MYERS,STEPHEN M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: ELECTROENCEPHALOGRAPHY -- EPILEPSY -- MODELS -- MONITORING -- SEIZURE -- TEMPORAL
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract: Platform Development for the Modulation of Epileptic Seizure Base on Interictal Spike Rate Epilepsy is one the most devastating neurological disorders in existence. Nearly one-third of patients have recurrent spontaneous temporal lobe seizures after pharmacological intervention. More often these patients are turning to types of adjunctive therapy such as vagus nerve stimulation. Current treatments based on vagus nerve stimulation use an open-loop simulation approach. Success of a closed-loop seizure prevention system would require identification of a measurable electroencephalogram (EEG) feature. Spike wave discharges often accompany epilepsy; however, the relationship between interictal spikes and seizure onset is largely unknown. Here, a method for recording, sorting interictal spikes and relating their occurrence with seizure onset in a rat model of temporal lobe epilepsy is presented. To determine this relationship first a study platform was created, which allowed for the chronic recording of highly sampled EEG signals. First a recording platform was created, and then using this system, EEGs containing spontaneous seizures were then recorded. Interictal spikes were extracted from these recordings and grouped into two major groups. When the number of spikes occurring per minute of recording was binned and plotted there was an increase in the ?ring rate of spikes before the occurrence of seizures. The change in spike rate also increased in the time just before an impending seizure. The results presented here show there is a temporal relationship between the rate of CA1 region interictal spikes and seizure onset in an animal model of temporal lobe epilepsy. With this information, a closed-loop seizure prevention system can be developed based on the modulation of interictal spikes.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by STEPHEN M MYERS.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Carney, Paul R.
Local: Co-adviser: Talathi, Sachin.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

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Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042553:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042553/00001

Material Information

Title: Platform Development for the Modulation of Epileptic Seizure Base on Interictal Spike Rate
Physical Description: 1 online resource (103 p.)
Language: english
Creator: MYERS,STEPHEN M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: ELECTROENCEPHALOGRAPHY -- EPILEPSY -- MODELS -- MONITORING -- SEIZURE -- TEMPORAL
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Platform Development for the Modulation of Epileptic Seizure Base on Interictal Spike Rate Epilepsy is one the most devastating neurological disorders in existence. Nearly one-third of patients have recurrent spontaneous temporal lobe seizures after pharmacological intervention. More often these patients are turning to types of adjunctive therapy such as vagus nerve stimulation. Current treatments based on vagus nerve stimulation use an open-loop simulation approach. Success of a closed-loop seizure prevention system would require identification of a measurable electroencephalogram (EEG) feature. Spike wave discharges often accompany epilepsy; however, the relationship between interictal spikes and seizure onset is largely unknown. Here, a method for recording, sorting interictal spikes and relating their occurrence with seizure onset in a rat model of temporal lobe epilepsy is presented. To determine this relationship first a study platform was created, which allowed for the chronic recording of highly sampled EEG signals. First a recording platform was created, and then using this system, EEGs containing spontaneous seizures were then recorded. Interictal spikes were extracted from these recordings and grouped into two major groups. When the number of spikes occurring per minute of recording was binned and plotted there was an increase in the ?ring rate of spikes before the occurrence of seizures. The change in spike rate also increased in the time just before an impending seizure. The results presented here show there is a temporal relationship between the rate of CA1 region interictal spikes and seizure onset in an animal model of temporal lobe epilepsy. With this information, a closed-loop seizure prevention system can be developed based on the modulation of interictal spikes.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by STEPHEN M MYERS.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Carney, Paul R.
Local: Co-adviser: Talathi, Sachin.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042553:00001


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PLA TFORMDEVELOPMENTFORTHEMODULATIONOFEPILEPTICSEIZURES BASEDONINTERICTALSPIKERATE By STEPHENM.MYERS ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 2011

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c 2011 StephenM.Myers 2

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T omywifeandparents 3

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A CKNOWLEDGMENTS Numerouspeoplehavehelpedmereachthispoint.Imustrstthankmywife, Renee.Theroadofbeingmarriedtoagraduatestudentcansometimesbealongand difcultone.Thankyouforyourpatiencewithmeonthisjourney.Youhavemadethe adventuremoreenjoyablethanitwouldhaveeverbeenhaditbeentravelledalone. EverydaywithyouisablessingthatIameternallythankfulfor.Secondly,Iwouldliketo thankmyparents.Youhavebeenamazingrolemodels.Iamthankfulforyoursupport andlove. Thelastveyearswouldofcoursenotbepossiblewithoutmymentorsandlab mates.Dr.Carney,thankyoutheuniqueopportunitytoworkinatranslationalresearch lab,wherebiomedicalengineersshouldbe.Thechancetotrulyseefrombenchto bedsideinactionissomethinganybiomedicalengineerwouldappreciate.Mylabmates, youhaveprovidedmewithmanygreattimes.Mydayswillneverbethesamewithout youinthem.Jason,thankyouforkeepingtheengineeringsideofmybrainentertained withourthoughtprovokingdiscussionsoverbowling.Rabia,youwerethesisterInever had,thankyouforkeepingmeorganizedandforknowingeverythingthatIdidn't. 4

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T ABLEOFCONTENTS page A CKNOWLEDGMENTS ..................................4 LISTOFTABLES ......................................7 LISTOFFIGURES .....................................8 ABSTRACT .........................................10 CHAPTER 1INTRODUCTION ...................................12 Motivation .......................................12 Terminology ......................................13 Spikes ....................................13 RacineScale .................................14 ProblemStatement ..................................14 Objective1 ..................................15 Objective2 ..................................15 Background ......................................15 EpilepsyBackground ............................15 TemporalLobeEpilepsy ..........................17 EEGBackground ..............................18 SpikePathology ...............................19 Contribution ......................................20 2MODELSOFEPILEPSY ..............................22 PurposeofEpilepsyModels .............................22 ModelCharacteristics ................................24 ModelSelection ...................................25 AnalysisoftheModelforSelfSustainingStatusEpilepticus ...........27 3RESEARCHMETHODOLOGY ...........................37 AnimalPreparation ..................................37 AnimalSurgeries ..............................37 Bipolarelectrodeimplantation .....................38 Microwireimplantation .........................40 EMGimplantation ...........................40 PostSurgeryRecovery ...........................41 StimulationProtocol .............................41 AnimalHousing ....................................42 RecordingChambers ............................42 EEGRecordings ...................................43 5

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4 PLATFORMDEVELOPMENT ............................48 RecordingSystems .................................48 ElectrodeConstructionandUse ..........................50 BipolarTwistElectrodes ...........................50 Microelectrodes ...............................50 5RELATIONSHIPBETWEENSPIKERATEANDSEIZURE ............56 Introduction ......................................56 Methods ........................................57 Analysis ........................................59 DiscussionandResults ...............................62 Conclusion ......................................64 6CONCLUSIONS ...................................78 FinalRemarks ................................78 APPENDIX AFURTHERCONSIDERATIONS ...........................80 ReducingElectrographicalNoise ..........................81 RecordingSetup ...................................81 DataConversion ...................................82 AdditionalDevices ..................................83 BADDITIONALEXPERIMENTS ...........................90 MethodsforHybridModel ..............................90 PreliminaryResultsforFutureExperiments ....................91 DirectStimulationtotheCA1 ........................91 SeizureInterventionWithDirectStimulation ...............91 VNSExperimentalSetup ..........................92 REFERENCES .......................................96 BIOGRAPHICALSKETCH ................................103 6

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LIST OFTABLES T able page 5-1 Percentageofpreictaldistributionsthatwerestaticallydifferentthaninterictal distributions ......................................71 7

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LIST OFFIGURES Figure page 1-1 Representativeplotofanactionpotential .....................21 1-2InterictalepileptiformdischargesasseeninrecordingsfromtherightCA1 (RCA1)andleftCA1(LCA1)depthelectrodes. ..................21 2-1TimelineforSEmodel ................................31 2-2Schematicofhowthebraintransitionsfromnormaltoepileptic .........31 2-3Histogramshowingwhenseizureoccurred ....................32 2-4Circadiannatureofseizureoccurrence ......................33 2-5Timedifferencebetweenseizures .........................34 2-6Histogramofseizureoccurrence ..........................35 2-7Seizurespergrade ..................................36 3-1Exposedskullpriortomarkingthecoordinatesforelectrodeplacement ....44 3-2Stereotaxicframe ..................................44 3-3PlacementofElectrodesrelativetoanatomicallandmarks ............45 3-4Viewofheadstageimmediatelypostsurgery ...................45 3-5Illustrationofheadstage ...............................46 3-6SchematicofthelocationofmicrowireelectrodesforrecordingfromtheCA1 andDG ........................................46 3-7EMGimplantationtechnique ............................47 4-1Screenshotofcustomsoftwarefordataconversion ...............52 4-2Biopolarelectrodes ..................................52 4-318ChannelOmneticsconnector ..........................53 4-4Jigformicrowireconstruction ............................53 4-5SetupforTDTsystem ................................54 4-6Stellatesetup .....................................55 5-1Dynamicstatesseeninepilepsy ..........................66 8

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5-2 Electrodesplacementofbipolarelectrodesandstimulatingelectrodeforchronic recording .......................................67 5-3TheprocesstakentoextractspikesfromEEGdata ................68 5-4Plotofspikerateperminutevstime ........................69 5-5Meanringratesperminute .............................70 5-6Percentchangebetweenwindows .........................72 5-7Averageslopesofpreictalandinterictalwindows .................73 5-8ROCCurveofpreictalandinterictalspikeratevalues ..............74 5-9Plotofspikesoveramultidayperiod ........................75 5-10Spikesoccurringperminutevstime ........................75 5-11Normalizedspikerateofspikes ...........................76 5-12Comparisonofthemeanspikerateduringnonseizuretimesofbothnonseizing butinjuredanimalsandseizinganimals ......................76 5-13Hemispherecomparisonofaverageringrate ...................77 A-1GrassConnections ..................................85 A-210-20electrodecongurationbreakoutbox ....................86 A-3Conversionsoftware .................................87 A-4Automatedratfeederthroughthedevelopmentprocess .............88 A-5Outputfromtrackingsoftware ............................89 B-1NumberofseizureinducedwithPTZ ........................94 B-2HeartratebeforeandduringVNStreatment ....................95 9

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Abstr actofDissertationPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulllmentofthe RequirementsfortheDegreeofDoctorofPhilosophy PLATFORMDEVELOPMENTFORTHEMODULATIONOFEPILEPTICSEIZURES BASEDONINTERICTALSPIKERATE By StephenM.Myers May2011 Chair:PaulR.Carney Cochair:SachinS.Talathi Major:BiomedicalEngineering Epilepsyisonethemostdevastatingneurologicaldisordersinexistence.Nearly one-thirdofpatientshaverecurrentspontaneoustemporallobeseizuresafterpharmacological intervention.Moreoftenthesepatientsareturningtotypesofadjunctivetherapysuch asvagusnervestimulation.Currenttreatmentsbasedonvagusnervestimulationuse anopen-loopsimulationapproach.Successofaclosed-loopseizurepreventionsystem wouldrequireidenticationofameasurableelectroencephalogram(EEG)feature. Spikewavedischargesoftenaccompanyepilepsy;however,therelationshipbetween interictalspikesandseizureonsetislargelyunknown.Here,amethodforrecording, sortinginterictalspikesandrelatingtheiroccurrencewithseizureonsetinaratmodelof temporallobeepilepsyispresented. Todeterminethisrelationshiprstastudyplatformwascreated,whichallowedfor thechronicrecordingofhighlysampledEEGsignals.Firstarecordingplatformwas created,andthenusingthissystem,EEGscontainingspontaneousseizureswerethen recorded.Interictalspikeswereextractedfromtheserecordingsandgroupedintotwo majorgroups.Whenthenumberofspikesoccurringperminuteofrecordingwasbinned andplottedtherewasanincreaseintheringrateofspikesbeforetheoccurrenceof seizures.Thechangeinspikeratealsoincreasedinthetimejustbeforeanimpending seizure. 10

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The resultspresentedhereshowthereisatemporalrelationshipbetweenthe rateofCA1regioninterictalspikesandseizureonsetinananimalmodeloftemporal lobeepilepsy.Withthisinformation,aclosed-loopseizurepreventionsystemcanbe developedbasedonthemodulationofinterictalspikes. 11

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CHAPTER 1 INTRODUCTION Motivation Epilepsyisoneofthemostcommonchronicneurologicaldiseasesintheworld [79].IntheUnitedStatesalone,itaffectsnearly1-5%ofthepopulation[ 23, 47].In additiontothese3millionpatientsthatlivewithepilepsy,10%ofthepopulationwill experienceaseizureduringtheirlifetime.ItisestimatedepilepsycostsAmericansover $15billioneachyear[ 24].Whilethereisnocureforepilepsy,thereareanumberof treatments.Unfortunately,forthosepatientsaffectedbyepilepsy,nearlyathirdofthem areunresponsivetothepharmacologicalinterventionandneverenterintoremission [75, 84].Despitethemostadvancedmedicalintervention(antiepilepticmedication, surgery,etc)10%ofpatientswillneverbefreefromseizures. Thecurrentfailureofourmedicalknowledgetotreatthesepatientseffectively, leavesalargeneedforimprovedepilepsytherapy.Notonlyareone-thirdofpatientsnot ndingtheresultstheyseek,butmanyofthosethatdoenterremissionhavetodealwith anumberofimpairingsideeffects. Devicedriveninterventionofepilepticseizurecontrol,isshapingitselftobea promisingareaofepilepsytreatment.Currentlytherearetwomajordevicebased interventions,vagusnervestimulatorsanddeepbrainstimulators.Todatethesedevices havelimitedfeedbackcontrol.Themajorproblemwithafeedbacksystemisidentifyinga suitablebiomarkerthatiscapableofdeterminingapointofmodulation. TocombattheshortcomingsinepilepsytreatmenttheNationalInstitutesof Healthhasdenedanumberofbenchmarkstomotivatetheprogressofepilepsy research.Thisprojectseekstoaddknowledgetotheeldinanumberofthesenewly setstandardsincluding: Toidentifyandvalidatebiomarkerstopredictthedevelopmentofepilepsyinat-risk individuals 12

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T ooptimizeexistingtherapiesanddevelopnewtherapiesandtechnologiesfor curingepilepsy. Developnewapproachesfortargetedtherapies. Initiateclinicaltrailsofnew,modied,andcombinationapproachesto enhancecurerates. Inconclusion,thisprojectseekstodeveloptechnology,techniques,andinitiatethe researchneededinordertocreateafullyautomatedseizuremodulationdevice. Terminology Spikes Theelectrographical spike canbeanambiguousterm.Forthispurposeitis importanttoexplicitlystatethedenitionthatwillbeused.Thetermspikeoftenmeans oneoftwogroupsofevents.Therst,actionpotentials(Figure 1-1 A),aretheallor nothingchangesinacell'selectricalmembranepotential.Thesearefastactingevents, whichtypicallyonlylastafewamilliseconds.Theseeventsaretheprimaryformof neuronalsignaling.Theactionpotentialrepresentstheactivityofasingleneuron.In contrastthesecondtypeofspike,populationspikes(Figure 1-1 B),arethesummed activityofsynchronouspostsynapticdepolarizations.Forthepurposeofthisseriesof studiesdealingwiththehippocampus,theprimarygeneratorsofelectrographicalactivity (seizures,spikes,etc)arisesfromthepyramidalandinterneuronsintheCA1andCA3. Thesepopulationspikesoccuronamuchlargertimescalethanactionpotentials,but typicallylastlessthan200milliseconds. Thereareanumberofpopulationsubtypes(e.g.interictalepileptiformdischarges, sharpwaves,afterdischarges).Betweenthesesubgroups,interictalepileptiform discharges(Figure 1-2)areassociatedwithepilepsyatahighenoughratetoused forthediagnosisofepilepsy[ 11].Thesortingmethodspresentedhereareunableto distinguishbetweenthedifferentsubtypesofspikes.Forthepurposeofthiswork,all thesesubtypeeventswillbethespikesofprimaryinterestwhendetectingevents,and 13

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will bethetypeofeventreferredtowhenthegeneralterm spike isused.Thesespikes musthavethefollowingquantitativeandqualitativecharacteristics[ 21]: 1.Mustbeparoxysmal. 2.Mustshowabruptchangeinpolarity. 3.Durationmustbelessthan200milliseconds. 4.Thespikemusthaveaphysiologicaleld. Additionally,thesespikesaretypicallyfollowedbyadeltaslowwave.Whenthese eventsareextractedfromEEGandsortedintogroups,theaboverequirementswillbe usedtoclassifyoneclusterashavinganidealspikeprole.Eachanimal'sidealspike willbethespikegroupfollowedthroughtheanalyzationphase. RacineScale Throughoutthiswork,thebehavioralseverityofseizureswillbegradedbased ontheRacineScale.TheRacineScale[ 74]isapopularscalefortheclassicationof epilepticseizuresinrats.Thescalerangesfrom0to5with5beingthemostsevere. Grade0 Afterdischargeswithnophysicalmanifestations. Grade1 Facialclonus. Grade2 Headnodding. Grade3 Forelimbclonus. Grade4 Bilateralforelimbclonuswithrearing. Grade5 Generalizedclonicconvulsionswithlossofbalance. ProblemStatement Thepurposeofthisbodyofworkistobuildafoundation,bothinmethodsand research,forthestudyofclosedloopseizuremodulation.Previousworkintheeld, focusesonacutestudiesofthebrain,becauseoftheinherentdifcultyoflongterm, highlysampledelectroencephalography(EEG)studies.Theworkalsoinvestigatesthe temporalrelationshipbetweenseizuresandEEGspikesforfutureuseasafeaturefor closedloopmodulation. 14

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Objective 1 Therstobjectiveofthisstudywastodevelopthemethodsandequipmentrequired tobuildasystemtotestaclosedloopseizuremodulationdevice.Thisprimarilyinvolves thecreationofmethodsthatwereusedforrecordinglongtermandhighlysampledEEG signals.Usingthesemethods,aseizuredatabasewasthencreated,whichprovideda resourcetoinvestigateEEGtimeseriesastheyprogressedtowardsseizure. Objective2 Thesecondaimofthestudywastotaketheseizuredatabasesandquantifythe relationshipbetweeninterictalepileptiformdischargesandseizures.Todatethese spikesarebelievedtobehighlycorrelatedtodiseasedtissue;however,theirtemporal correlationtoimpendingseizuresisstillhighlydebatable. Background EpilepsyBackground Epilepsyisoneofthemostcommonchronicneurologicaldiseases.Itaffects nearly1%oftheworld'spopulation[ 21, 35, 78]withnearlyathirdofepilepticpatients nevergainingfullremissionofseizures.Epilepsyischaracterizedbyitsrecurrent, spontaneousseizures[ 78].Thelargenumberofaffectedpatientscanbelinkedtothe disease'smanycauses,whichincludebothgeneticdefectsandphysicalinsults.This makesepilepsynotasingledisease,butratheraclassofsymptomsthatarisesfroma numberofpathologicalstates.Thisinitselfmakesepilepsyadifcultdiseasetostudy. Inalmostallcasestherecurrent,spontaneousseizureisamainstayofanepileptic disorder.Thespontaneousnatureoftheseizurealsomakesepilepsyadifcultdisease tostudy.Patientsarerelativelynormalwhennotseizing,butseizuresthemselvescan haveextrememorbidity.Thetransientnatureoftheeventscorrelatestofewoccurringin aresearchorhospitalsetting. Seizurescanbecategorizedintomanytypes;however,theytypicallyresultin synchronizationofalargenumberofneurons,whichresultsfromanimbalanceof 15

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inhibitor yandexcitatoryneurons[ 7].Theseizuresresultingfromtheseimbalancesare classiedintotwomajorcategorized,partialandgeneralized[ 13, 27].Duringpartial seizures,theseizurehasaprimaryfocusthatgivesrisetotheseizure.Conversely, generalizedseizuresencompasstheentirecortex.Themanymanifestationsofepilepsy makeitahighlydynamicdisease,withseemlyrandomtransitionsbetweenseizureand normalstates. Theunpredictabilityofepilepsyisverydebilitatingforthoseitaffects.The paroxysmalnatureofepilepsynotonlycausespathologicalandneuropsychiatricissues, butalsosocial.Theoccurrenceofseizureswithoutwarningmakesepilepsytheleading causeofneuropsychiatricdisabilityworldwide[ 21].Studieshaveshownthesociological costofepilepsyalsoaffectsthepatient'slovedonesaspatientsexperiencetremendous emotional,nancial,self-esteem,andfamilyissues[ 10].Thesocialeffects,combined withdecreasesincognitivefunction,adverseeffectsofanti-epilepticmedicationsand theshearunpredictabilityofseizuresallculminateinanimpairedqualityoflife. Todate,themostprolictreatmentforepilepsyistheuseofantiepilepticdrugs (AED).Itisimportanttonote,alldrugsclassiedasanAEDarenotantiepilepticbut ratherantiseizuremedication.Noneofthesedrugsarecapableofalteringthecourse ofthediseasethatisepilepsy[ 72],butratheronlyservertosuppresstheoccurrenceof seizures.IfapatientwasresponsivetoAEDmedication,suspensionofthemedication wouldresultinthereappearanceoftheseizures.ThedevelopmentofAEDsisoften slowwithlongperiodsofnonewdrugstomarketbeingseen[ 49],againgivingrise fortheneedofnewtreatments.AEDsoperateunderthreebasicprinciples:(1)inhibit excitation,(2)enhanceinhibition,or(3)modifycellexcitabilitybythemodulationof voltagedependentionchannels[ 72]. Unfortunately,fornearlyathirdofthepatientssufferingwithepilepsy,theywill continuetohavespontaneousseizuresdespitethebestmedicinehastoofferinterms ofpharmacologicalintervention.Manyofthesepatientsareseekingnewformsof 16

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treatment. Forthesepatientssurgicalresectionoftheepilepticfocus,oravagus nervestimulatormaybetheironlyhope.Whiletheuseofsurgicalinterventionforthe treatmentofepilepsyisnotanewconcept[ 17],itisstillthoughtofasradicalbymost patients.Ithasbeenreportedthatlessthan2%ofpatientsthatarecandidatesfor surgicalresectionhavethesurgery[ 20].Ifpatientsdonothaveaseizurefocusorhave afocusthatisanareathatcannotberesectedthentheymustseekanotherformof augmentedtherapy. Thenewestaugmentedtherapyforthesepatientsisvagusnervestimulation.Vagus nervestimulationwasrstusedforthecontrolofseizuresin1988[ 69]andbecameFDA approvedin1997.Thetreatmentconsistsofimplantingapacemakerlikedeviceunder thecollarbone.Leadsarethenconnectedtotheleftvagusnerve.Thevagusnervehas widespreadinhibitoryprojectionstomanypartsofthebrain[ 58, 60, 76, 77].Studies haveshownvagusnervestimulationhaspositiveresults,withoverhalfofpatients seeinga50%decreaseinseizurefrequency[ 26]. TemporalLobeEpilepsy Themostadvancedtherapiesstillleavemillionsofepilepticpatientswithreoccurring seizures.Evenwiththeseadvances,thenumberofpatientsstillunresponsiveto treatmentshowsnewtherapiesarestilldesperatelyneeded.Ofparticularinterestfor thisstudyistemporallobeepilepsy(TLE).TLEisaparticularlydevastatingincarnation ofepilepsy.Itoftenbeginsinchildhoodandcontinuesthroughoutthepatient'slife.Of thepatientswithTLE,fewerthan20%willbecomecompletelyseizurefree[ 34, 42, 46]. ThisstudychoosestostudyTLEbecauseitiswidespreadamongtheepileptic populationandisoneoftheformsofepilepsythatismostcommonlyresistantto anti-epilepticdrugs[ 8]. TLEoccursinthestructuresthatcomprisethetemporallobe,specicallythe hippocampus,parahippocampalgyrusandamygdala[ 1].TLEoccurswhentheseareas experiencedegenerationandsclerosis[ 28].TLEissubdividedbasedontheregionit 17

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aff ectsintomesialtemporallobeepilepsy(MTLE)andlateraltemporallobeepilepsy (LTLE).OftheseMTLEisthemostcommon,andtheformthatwillbemodeledand studiedthroughouttheseexperiments.PatientswithMTLEsufferfrompartialseizures, whichcausecognitive,motorandsensoryimpairments.Severepartialseizurescan causealteredmentalstatesandconvulsivemovementseizures.Generalseizures aremoreseverethanpartialseizureandareassociatedwithalteredconsciousand tonic-clonicmotorseizures. EEGBackground Thebodyandelectricityhavebeenlinkedsincethelate18 th century,whenLuigi Galvanidiscoveredelectricitycouldcauseamusclecontractioninafrog'sleg.Nearly 100yearslateritwasdiscoveredthatstimulationofthebody'ssensorysystemresulted inelectricalactivityinthebrain.Thenin1912,therstelectrographicalseizurewas witnessed.In1929,HansBergerrecordedtherstscalpEEG,changingthediagnosis andtreatmentofepilepsyforever. TheEEGisgeneratedfromthesummationofsynapticpotentials(excitatory postsynapticpotentialsandinhibitorypostsynapticpotentials).Asionsowthroughthe neuron'spermeablemembrane,chargeowsintheoppositedirection.Thusaregion oftheneuronischargedcomparedtotherestofthecell.Thisseparationofcharge istermedadipole,anditistheprojectionofthedipoletotheEEGelectrodewhichis recordedintheEEG.Theplacementoftheelectrodecanhaveagreateffectonthe waveformwhichitrecords.Thescalpelectrodesthataretypicallyusedinclinicalsetting primarilyreceivesignalsfromthecortexdirectlybeneaththem.Sincethisstudyseeksto understandepilepsyintissuedeepwithinthebrain,intracranialelectrodesarerequired tobeplacedinorneartheseregionsofinterest. TheEEGisuniqueinthatitcanprovideahighleveloftemporalresolution, samplescanberecordedquicklyenoughtocapturethemostbasicelementofneuronal communication-theactionpotential,anditcanalsoprovideahighamountofspatial 18

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resolution throughtheadditionofmultipleelectrodes.Theelectrodescanbeplaced superciallyorintracranial.IntracranialelectrodesintroduceanewsubclassofEEG calledelectrocorticography(ECoG).ECoGelectrodescanbeplacedinspecicregions ofinterestofthebrain.Beingclosertothesourceoftheelectrographicalgeneration, theelectrodesrecordsignals10timesgreaterinamplitudecomparedtothoseseenin scalprecordings[ 33].Theserecordingshavebeenshowntorecordthesameelectrical potentialsasseeninscalpelectrodes[ 82];however,higherfrequenciesareseenwith ECoGrecordings[ 33].Additionally,theuseofinvasiveECoGelectrodesallowsfora smallerpopulationofneuronstobemonitored[ 83].Thenumber,typeandplacementof electrodesmustbedictatedbytheexperimentalquestion. SpikePathology Thenormalandabnormalbrainiscapableofgeneratinganumberofuniqueevents [33].Onesubclassofthiselectrophysiologicalactivityisthepopulationspike(spike). Whilenotallspikesarepathological[ 11],interictalspikesareauniqueformofspike correlatedtoepilepsy[ 11, 21, 39].Theircorrelationtoepilepsyislargelyundebated; however,theirtemporalrelationshiptoseizuresishighlydebatable.Currently,studies havenegative[ 1, 85],no[ 11, 31, 32]orunclear[ 2, 80]relationshipbetweenseizuresand spikes. Thespikeistheresultofasynchronousexcitationofapopulationofepileptic neurons.Individuallytheseneuronsgenerateactionpotentials.Ifthereisenough synchronybetweentheneurons,thesummationoftheirdepolarizationsislarge enoughtobedetectedbyextracellularrecordingelectrodes.Anumberofstudies haveshownthatspikesandseizuresaregeneratedthroughdifferentcellularand networkmechanisms[ 11];however,spikeshavebeenshowntoarisefromthedamaged areasofthebrainknownastheirritativezone[ 30 53].Thespikegeneratingzonedoes nothavetobetheseizurefocus,buttheyaregenerallyinthesamehemisphere.The correlationbetweenseizurefocusandspikepresenceishighenoughtousespikes 19

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as anelectrographicalmarkerfortheepileptogeniczone.Thesespikesareseen inanumberofmodelsofepilepsy,andinanumberofregionsofthebraininthese models.Whilethemethodspresentedinthisstudyarenotabletodistinguishbetween pathologicalspiketypes,pathologicalspikesdodifferfromnormalspikesbyhavinga moreregularshape,higheramplitude,andshorterduration[ 11]. Thehippocampus,andspecicallytheCA3,hasbeenshowntobeagenerator ofspikes[ 3, 9, 40].Sincethepopulationspikesarisingfromthetrisynapticloophave beenshowntobeinhibitoryinnatureandimportantinminimizingseizureoccurrence [4, 18, 44],thisstudywillrecordspikesfromthehippocampusinordertotrulydecipher therelationshipbetweenseizuresandspikes. Todate,thestudyofspikeshasbeenhinderedbyseveralkeyissues.Primarily amongthemareanon-uniformdenitionofspikes,lackofcontinuousrecordingsto provideatruewindowintotherelationship,anddifferencesbetweencorticalinterictal spikesandinterictalspikesrecordedataseizuresfocus[ 30]. Contribution Successofaclosed-loopseizurepreventionsystemwouldrequireidentication ofameasurableEEGfeaturethathasadirectcorrelationtoseizures.Spikesoften accompanyepilepsy;however,therelationshipbetweenspikesandseizureonsetis largelyunknownandhighlydebated.Hereamethodforrecordinghighlysampled,long termEEGsignalsandthenshowingthetemporalcorrelationbetweenseizuresand spikesispresented.Thisinformationcanthenbeusedastherststepsinbuilding aclosedloopsystemforthecontrolofepilepticseizurebasedoninterictalspike modulation. 20

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A ActionPotential B PopulationSpike Figure1-1.Representativeschematicplotofanactionpotential(A)andapopulation spike(B).Notethevastdifferenceinthescalebetweenthetwoplots. Figure 1-2.InterictalepileptiformdischargesasseeninrecordingsfromtherightCA1 (RCA1)andleftCA1(LCA1)depthelectrodes. 21

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CHAPTER 2 MODELSOFEPILEPSY PurposeofEpilepsyModels Thetransientnatureofepilepsymakesitadifcultdiseasetostudy.Theaverage epilepticpatientis,forallintentsandpurposes,normalwhennotexperiencinga seizure.Thespontaneityoftheepisodesmeanspatientswouldhavetobemonitored continuouslyforlongperiodsoftimetogarnermeaningfulresults.Inadditiontothe highcostassociatedwithhumanstudies,thereareanumberofethicalquestionsa researchermustface.Amajorconcernisthebalanceofgoodforthepatientvsthe goodofthegreatercollectivewholetheresearchmightyield.Theseconcernslessened byusinganimalmodels.Animalmodelsallowresearcherstogreatlyincreasetheir participantpoolwithouthavingtoinvolvehumanpatients.Supplementingthebenet ofcostreduction,animalmodelsoftenallowforamorethoroughstudyofadisease state.Animalmodelsallowforamoreinvasivestudyoftheepilepticbrain.When investigatingwithananimalmodel,aninvestigatorcanstudycellularmechanisms andotheraspectsofthebrainthatcouldneverbestudiedinahuman.Inadiseaseas dynamicasepilepsy,therehavebeenstudiesshowingthatdietandenvironmentcan haveaneffectonseizures[ 15, 25].Theuseofanimalsalsoallowsformanyofvariables tobecontrolled. Epilepsyisacomplicatedanddynamicdisease.Forthisreason,anumberof differentmodelsexist,whichallowsforthestudyofvariousaspectsoftheepileptic state.Therearetwoprimarygroupsofmodel[ 19].Therstistheacutemodel.In thesemodelsofepilepsy,someaspectofepilepsyisstudiedinarelativelynormal brain.Seizuresaremanifestinanacutemanorthroughoneofanumberofstimulants. Thesestimulantscanbepharmacological,electrical,oraudiogenicinnature[ 70, 71]. Whenexposedtothestimulant,anacuteictaleventisgenerated.Whilethereisoften adisconnectbetweentheseseizuresandseizuresthatwouldbeseeninepileptic 22

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patients ,theacutemodeldoesallowforthestudyofanumberofuniquefeaturesofthe epilepticbrain.Primaryamongsttheseisthestudyofthecellularandmolecularbasisof aseizure[ 71].Throughtheuseofthesemodels,investigatorshavebeenabletostudy changesabouttheintrinsicpropertiesofneurons. Thesecondtypeofmodelisthechronicmodelofepilepsy.Themostimportant aspectofthechronicmodelisitsabilitytoletinvestigatorsstudythelatentandinterictal periods.Thisprovidestheopportunitytostudymoreclinicallyrelevantmechanisms.As mentionedinsection 1,temporallobeepilepsyisaparticularlydevastatingincarnation ofepilepsybecauseofitsresistanttomanymodernepilepsytreatments.Chronic modelsaremostoftenusedtostudythisformofepilepsy[ 86].Thevarioustypesof chronicmodelscangeneratedamageinthelimbicstructuresofthebrainsimilarto sclerosisseeninhumanpatientssufferingfromMTLE.Onetypeofchronicmodelis thekindlingmodel.Inthismodel,stimulatingelectrodesareplacedintotheamygdala andstimulatedfor2secondstwiceadayuntilgrade5seizuresareseen[ 29, 57].These repetitivestimulationswilldamagethebrain,eventuallycausingtherattoseizewhen stimulatedwithanotherpulseofelectricity.Thisisapopularmodelforthestudyof newdrugsasit(1)causesondemandseizuresand(2)causesseizuresthatarevery repeatablefromtrialtotrial.Asecondsubclassofchronicmodelistheselfsustaining statusepilepticus(SSSE)model.Statuscanbeinducedthrougheithermedication orelectricalstimulationofthebrain.Whenusingelectricalstimulation,stimulating electrodesareplacedintotheventralhippocampus.Inthismodelthehippocampus isstimulatedforroughly1.5hours.Thisinducesstatusepilepticsintheanimal,which continuesafterthestimulationhasbeenstoppedandisthustermedselfsustaining statusepileptics.Afterrecovering,theanimalwilldevelopseizuresfourtoeightweeks poststimulus.Theseseizures,liketheirhumanequivalent,arespontaneous. 23

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Model Characteristics Choosingthepropermodeldependsontheexperimentstobedoneandthe particularfacetofepilepsythatonewishestostudy.Theelectroencephalogramis themostwidelyuseddeviceforthediagnosisandstudyofepilepsy.Therefore,when studyingmanyclinicalquestionsitisimportantthatamodelgeneratesseizuresthat areelectrographicallysimilartotheirhumancounterparts.Forthisseriesofstudies, electrographicalbiomarkersareveryimportant.Thus,itiscriticaltopickamodelthat notonlyproducesseizuresbutcanalsoproduceelectrographicalevents,suchasspikes andhighfrequencyoscillations,thatmightbecapableofactingasabiomarkerfor seizures.Chronicmodelsalsoyieldtheinvestigatortheuniqueopportunitytostudy theentirediseasestateandnotjusttheseizureitself.Thedifferentstagestheepileptic evolutionare: LatentPeriod-thetimefromtheinitialinsultthatcausesepilepsytotherst seizure. Interictal-thetimebetweenseizures. Preictal-thetimejustpriortoaseizure. Postictal-thetimejustafteraseizure. Preictalandpostictalarenebuloustermsbecausethetransitionintoandoutofa seizurecanbeslowanddifculttodetect.Forthispurposewhenanalyzingdatainthis study,thesevariableswillbesettoconcretetimelimitsassupportedbytheliteratureon thesubject. IntheEEG,therearealsoseveralothercharacteristicsthatcanbemeasured.The mostimportantoftheseistheseizureitself.Seizurescanbebrokendownintotwo majorcategories,partialandgeneralized.Animalbehaviorisalsoanothermetricthat canmonitored.Modelsmayeitherproducefullonsetseizures,whichalwayspresent physicallyasafullbodyclonusoftheanimals,oraspartialseizures.Theadventof noninvasiveimagingtechniques(e.g.MRI,fMRI)hasincreasedtheinterestinmodels 24

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with accuraterepresentationofthestructuralchangesthatareassociatedwithepilepsy. StructuralchangesareoftenofspecialinteresttostudiesinvolvingMTLEbecausethose itaffectsoftenexperiencelesionsinthebrain. ModelSelection Forthepurposeofthesestudiesamodelneedstobechosenthatis: Chronic Producesspontaneousseizureswithenoughinterictaltimetoallowtheanimal modeltoreturntobaselinebetweenseizures. CloselymimictheMTLEconditioninstructuralchangesandresponseto treatment. Afterreviewingtheliteratureitwasdeterminetoworkwiththeaselfsustaining statusepilepticusmodel.Thistypeofmodelwaschosenbecauseitsdamagetothe hippocampusmimicsthefocalpathologiesinpatientswithMTLE[ 5].Thefullmethod ofinducingananimalintoselfsustainingstatusepilepticuswillbedescribedlaterin section 3,butageneralmechanismandoverviewwillbediscussedhere. Pathologically,themodelrecreatestheseverehippocampalsclerosiscausedbythe hilarandCA1neuronalloss[ 54, 55].Studieshaveshownthatfollowingtheelectrical insult,thereisareductioninGABA A receptormediatedsynapticinhibition[ 51, 52]. SinceGABAisthemaininhibitorinthecentralnervoussystem[ 64]thelackofinhibition thencausesthepyramidalcellsinthatCA1regionofthehippocampustobecome overlyexcitable.ThestimulationappearstobeindiscriminateinitsreductionofGABA receptors,asstudieshaveshownitalsoreducesGABA B receptors[ 52].Thereduction ininhibitionseemstoplayamajorroleinthedevelopmentofseizures;however,the completemechanismforseizuredevelopmentisnotyetcompletelyunderstood. Themajorbenetforthisseriesofstudiesistheelectrographicmorphologythe modelgenerates.Theseizuresandtheirresponsetocurrentepilepsytreatments areverysimilartothoseseeninhumans.LikehumanMTLE,theseizureshavea 25

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similar focusandaretypicallypharmacoresistant.Themodelgivesasufcientlylong latentperiod(Figure 2-1),whichisbenecialforstudiesthatwishtodevelopcurative treatmentsforepilepsyorwishtounderstandhowepilepsydevelops.Studieshave shownseizurescanbeginbetween7daysand3monthsafterstatusepilepticus[ 56]. Theseizuresproducedbythismodelareidealtostudybecausetheyalsomimicthe severityandinterictaltimeofMTLE. Statusepilepticusisdenedasastateofcontinuousseizureactivitylastingatleast 30minutes[ 48].Thecontinuoushippocampalmodelofselfsustainingstatusepileptics thatwasusedinthesestudiesgeneratesstatusepilepticusbyelectricalstimulation oftheCA3portionofthehippocampus.Theelectricalstimulationofthehippocampus continuesfor30to90minutes.Afterbeingdiscontinued,iftheanimalcontinuesto seizethentheanimalissaidtobeastateofselfsustainingstatusepilepticus.The amplitudeofelectricalstimulationwasinitiallychosenbasedontheworkdonein kindlingmodelsofepilepsy.However,thecurrentstimulationwaslaterrenedasbeing twicethecurrentrequired,onaverage,togenerateafterdischargesintheanimal[ 56]. Foranormalanimal,afterdischargesbeingat150 A.Ifananimaldoesnotexperience afterdischargesuntil300 Athenthelikelihoodtheanimalwillenterintoselfsustaining statusislow. Hybridmodelofstatusepilepticus .Themaindrawbacktoselectingtheself sustainingstatusepilepticusmodeliswhentryingtoinvestigatetherapeuticintervention. Whiledeemedbenecialformostaspectsofthestudy,thespontaneousseizurecauses aproblemwheninvestigatingvariousinterventionschemes.Withseizuresoccurring betweenoneandseventimesaweek,itwouldtakeaconsiderableamountoftimeto generateenoughdatatostatisticallyproveatherapy'sbenets.Inlightofthisdecit, severalexperimentswereperformedinordertocreateahybridmodel.Thepurposeof thishybridmodelistocreateamodelwithallthecharacteristicsoftheselfsustaining SEmodelyethavethecapabilitytoinitiateanondemandseizure.Amodelthatsimply 26

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creates ondemandseizureswasnotusedbecausetheygenerateseizuresthatarenot similartothosefoundintemporallobeepilepsy. Thetheorybehindthismodelisthatwhentheanimalisinitsinterictalstateitis predisposedtohavingaseizure.Theimpetushasbeenseverelyheightenedandonlya slightperturbationinthebraincouldcausethebraintocrossathresholdintoaseizure (seeFigure 2-2).Forthehybridmodel,Pentylenetetrazol(PTZ)wasthechosenstimulus toperturbthebrainintoseizure.PTZisacommonproconvulsantthathasbeenshown toworkinawiderangeofanimals.PTZworksbyactingontheGABAreceptorsinthe braintoimpairGABA-mediatedinhibition[ 66].PTZcanbeusedtocreatebothacute andchronicmodelsofepilepsy.Inacutesituations,PTZisusedtogenerategeneralized seizuresforthestudyofantiepilepticdrugs[ 43].Afterthedeliveryofalargebolusof PTZ,seizurestypicallybeginlessthan30minutesafterinjection[ 71].Theseizures generatedcanincluded:freezing,myclonictwitches,clonicseizures,andtonic-clonic seizures[ 71].Basedtothedesiredreaction,thedosagecanbetitratedtogeneratemild, electrographiconlyseizures,tosevere,tonic-clonicseizures.Whenusedtocreatea chronicmodel,whetherthroughkindlingortheinductionofstatusepilepticus[ 63],PTZ createsgeneralizedtonic-clonicseizures.TogenerateachronicPTZmodelofepilepsy, roughlyhalfthedoes(or40mg/kg)isgivenascomparedtotheamountadministeredfor anacutestudyoverseveraldays[ 36]. AnalysisoftheModelforSelfSustainingStatusEpilepticus AprimarythrustofthisbodyofworkwastogenerateEEGdatacontaining spontaneouslyoccurringseizuresinordertocharacterizeseizuredynamicsfrom longterm,chronicrecordings.Theprimarymodelusedintheseexperimentswasthe selfsustainingstatusepilepticusmodel.Intotal,nearly900spontaneousseizures havebeenrecordedinthepastveyears.Theseseizuresincludemultichannel,highly sample(1000Hz)recordings,withtimelockedEEG.Todatenotmuchworkhasbeen 27

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done onthelongtermseizurestatisticsoftheSSSEmodelastheywouldapplytoa studyonseizureprediction. Thisstudyseekstoinvestigateseizureoccurrence(thetimeofdayaseizureis mostlikelytooccur),seizureinterval,andthedistributionofseizuregrade.Usingthelog lesgeneratedbytherecordingsystems,eachseizure'soccurrencewasplottedonto a24hourday.Ahistogramwasthenperformedonthisdatasettogivethenumberof seizureperhouroftheday(Figure 2-3).InFigure 2-3 (n=699seizures),thelightcycle informationisshownwiththeshadedbackground.Inhumans,itisknownthatseizures aremorelikelytooccurwhenispatientisjustenteringorexitingasleepstate.Rats arenocturnal,thereforethepatternofseizureoccurrenceshouldbeinvertedfortheir invertedsleepcycle.Theanimalsshowedthehighestseizureratesinthehoursjust beforethelightsturnedoff.Thiswouldequatetothenalstagesoftheirsleepcycleand correlatestowhatisseeninhumanpatients.Usingacosinortest,thedatainFigure 2-3 wasttoacircadianrhythm(p.001)(Figure 2-4). Becauseofitsspontaneousnature,thefrequencyofthismodel'sseizureshasnot beenstudiedindetail.Ifthemodelcontainsadominatefrequencyofseizureinterval, thenwhentestsaremadetocompareafullyclosedloopseizurecontrollerwiththe standard,tonicallyringseizurecontrollersusedintoday'sVNSdevices,thetonicring mayhaveanadvantageinsuchasituationthatwouldneedtobeaccountedfor. Aftertherecordingsweremade,animals(n=4)thathadmorethan10seizures wereusedtodeterminethefrequencyanddistributionofseizureseverity.Seizure interval(SI)timeswerecalculatedforalltheseizuresgeneratedbytheseanimals (SI = t (seizure n +1 ) t (seizure n )).Figure 2-5 showsnormalizedtimeintervalbetween neighboringseizures.Themediantimeintervalvariedhighlybetweenanimals,but thepurposeofthisstudywastodetermineif,oncetheintervalstabilized,therewasa seizurefrequencymostoftenencountered.Itwasdeterminedtonormalizetheintervals toseeifacommonfrequencypresenteditselfwhencomparingvariousanimals.Ifthe 28

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results wereconsistentthenthisnormalizedaveragecouldbeusedtoestimatethenext seizureoccurrence.Figure 2-5 A-Cshowanimalsinwhichrecordingsbeganshortly afterthecompletionofthemodelinduction.Doingsoallowedforthecaptureoftherst seizure.Thegureshowseachanimalinitiallystartedoutwithahighseizureinterval beforeitquicklyleveledoffafterseveralseizures.TheanimalshowninFigure 2-5 D doesnotcontainthisatliningbecauseitsrecordingsdidnotbeinguntilonemonthafter theinductionofthemodel.Allfouranimalsshowtheprogressiontowardsasteadystate ofseizureinterval. Plottingthissamedataasahistogramoftheseizureintervals(Figure 2-6),it becomeapparenttheseizuresarenotrandomlydistributed.Theanimalsshowed aconsistentaverageseizureinterval(ASI)betweenanimals(0.1339 0.03377, 0.1305 0.01103,0.07060 0.009926,and0.1461 0.006867).Comparingtheseizure intervalsonlyoneanimalhadameanthatwassignicantlydifferentthetheother animals. Thedistributionofseizuregradewasalsoimportanttounderstand.Humansalso experiencearangeofseizuresgradesanditwasimportanttodetermineifarangeof seizuregradeswerepresentintherecordings.Seizuregradeswerescoredusingthe Racinescale.Plottingthenumberofeachseizuregradefound,itwasdiscoveredthat highergradeseizuresaremorelikelytooccur(Figure 2-7).Astheinter-seizureinterval reachedasteadyrate,theanimalalsosettledonaseizureseveritythatwouldcomprise mostofitsseizures.Earlyseizuresweregenerallygrade2or3seizureswhileindividual animalswouldplateauandgeneratepredominatelygrade4or5seizures. Forthisstudy,43ratsenteredthestudyandprogressedthroughsurgeryandmodel induction.All43animalssurvivedsurgeryandmodelinduction.Ofthose43animals only16(37%)remainedinthestudytothepointofinitialEEGrecordings(1monthpost modelinduction).Themainreasonforremovalfromthestudywasduetodislodgment oftheheadstagecontainingtheelectrodes.Ofthe16animalsthatwererecordedand 29

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screened forseizureonly9producedrecordedseizures.The7thatdidnotseizefellinto twogroups,therst,andmostcommon,wereanimalsthatunderthestressoftheirrst seizuredislodgedtheirheadstage.Thesmallersubset(n=3)wereanimalsthatnever seizedafterseveralweeksofcontinuousrecordings.Effectively,1in5animals(20.9%) completedthestudywithspontaneousseizures. 30

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Figure 2-1.Timelineforselfsustainingstatusepilepticusmodelofepilepsy.Onday0 surgeryisperformed.Afterallowingtheanimaltorecoverforoneweek baselinerecordingsbegin.OneweeklaterselfsustainingSEisinducedin theanimal.Roughly4weekslaterspontaneousseizureswillbegin. Figure 2-2.ItwashypothesizedthatSEputstheanimalinahyperexcitedstate.Insuch astateitiseasierforthebraintotransitionintoseizure.Thiscartoon schematicshowshowthebrainbecomesmorepronetoseizuresasits normalstatenearsthethresholdforseizures. 31

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Figure 2-3.Histogramshowingwhenseizureoccurredforallanimals(n=699).Thegray backgroundindicateswhenthelightswereoffintherecordingsarea.The occurrenceofseizureswasshowntobecircadianinnature.Theanimals aremorelikelytoexperienceaseizurejustpriortothelightsoutconditionin thehousingunits. 32

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Figure 2-4.Plotshowingoccurrenceofseizuresduringa24hourday.Usingacosinor test,thesignalwasshowntobecircadian(p < 0.001,n=699seizures) 33

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Figure 2-5.Plotsrepresentingthenormalizedtimedifferencebetweenneighboring seizures.Withtimetheintervalbetweenseizureslevelstoarelatively constantrate.AnimalsA-CwererecordedimmediatelyafterSEinduction andthereforetheirrstseizureswerecaptured.AnimalDdidnotbegin recordingsuntil4weekspostSEinduction,andthereforeitsrstseizures werenotrecorded.Thisiswhyitsseizureintervalhasalreadyreached steadystate. 34

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Figure 2-6.Afternormalizingthehistogramsofseizureintervalbothintermsofinterval andcountperinterval,thehistogramsfromalltheanimalswereaveraged together.Whileactualseizureintervalwasvariedamonganimals,whenthe intervalwasnormalized,allanimalsshowedtohaveasimilarclusteringof interseizureintervals. 35

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Figure 2-7.Numberofseizuresineachgrade.Inthestudiedanimals,highgrade seizuresweremorelikelytooccurthanlowgradeseizures. 36

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CHAPTER 3 RESEARCHMETHODOLOGY AnimalPreparation Allexperimentswereperformedon2monthold,maleSpragueDawleyrats weighingbetween220-265grams.Allexperimentsandprocedureswereapproved bytheInstitutionalAnimalCareandUseCommitteeoftheUniversityofFlorida. AnimalSurgeries Initialsurgerypreparationforallsurgeriesfollowthesamegeneralmethods. Animalsarebroughttothesurgeryroom.Theroomwassegregatedintothreedistinct areas:surgerypreparationarea,surgeryarea,andrecoveryarea.Inthepreparation areatheanimalwasplacedinananesthesiainductionboxandexposedto4% isouraneatarateof1Lperminute.Onceinductionwascompletetheanimalwas removedfromtheinductionbox.Isouranewasthenreducedto1%at0.6Lperminute anddeliveredthroughanosecone.20mgofxylazinewasadministeredsubcutaneously toactastheprimaryanalgesicforthedurationofthesurgery.Isouraneadministration wascontinuedthroughoutthesurgerytosmooththeeffectsofthexylazine.The animal'stoepinchreexwascheckedtoensuretheanimalwascorrectlysedatedbefore thesurgeryproceeded.Oncecompletelyunresponsive,thetopoftheanimal'shead wasshaved.Theheadwasthenchemicallysterilizedwithanalternatingbathofiodine andalcohol.Fromheretheanimalwasmovedtothesurgeryareaandplacedina stereotaxicframe.Astereotaxicframewasusedinconjunctionwitharatatlas[ 68]to preciselypositionelectrodes. Thetopoftheskullwasexposedviaamidsagittalincisionthatextendedfrom betweentheeyestothetoearlevel.Theskull'sfasciawasseparatedfromtheskullto exposetheanatomicallandmarks,lambdaandbregma.Fourcurvedhalstedmosquito forcepswereusedtoretracttheskinandfascia,allowingforampleworkspaceatopthe skull(Figure 3-1).Aperoxidewashwasappliedtothesurfaceoftheskulltoremoveany 37

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remaining softtissue.Thestereotaxicarmwasusedtodenotethebregmalandmarkas (0,0).Itwasfromthispositionthatallfuturepositionswerecalculated. Bipolarelectrodeimplantation Thecommonsurgeryforbipolarelectrodeimplantationconsistsofcontralateral electrodeplacementaccompaniedbytheimplantationofagroundandreferencescrew electrode.Thecontralateralbipolarelectrodescanbeplacedattheinvestigatorsdesired location.Forthepurposeofthisstudy,thebipolarelectrodeswereplacedintheCA1of eachhippocampus. Oncetheskullhasbeenexposedandallbleedingstopped,electrodeplacement canbegin.Whenusingastereotaxicinstrumentwithitsarmontheleft(Figure 3-2), electrodeplacementshouldproceededtoptobottomandrighttoleft.Thismethodology shouldbefollowedtoinsurethestereotaxicarmdoesnotinterferewithlaterplacement ofelectrodes. Usingthestereotaxicarm,thefollowingpositionsweremarkedontheskullsurface: 1.RightCA1:AP-4.3mm,Lateral2mm 2.LeftCA1:AP-4.3mm,Lateral-2mm 3.GroundElectrode:AP2mm 4.ReferenceElectrode:AP-6mm Usinga0-80stainlessstealdrillbit,pilotholesweredrilledforthegroundand referenceelectrodesattheirrespectivelocationswithahanddrill.Additionalholesfor anchoringscrewswerethenalsomade(Figure 3-3).Theseholesweredrilled3mm anteriortotherecordingelectrodes.Workingfromfronttoback,thegroundelectrode wasscrewedintoitspilothole.Aproperscrewdepthisonethatisdeepenoughto securethescrewtotheskullwithoutcausingdamagetothebrain.Theelectrode wireshouldthenbebentoutoftheworkingsurgicaleld.Next,usingatrephinebit andDremelhandtool,a2mmwidesectionofskullwasremovedatthelocations markedforthebipolarelectrodes.Abipolarelectrodewasplacedinthestereotaxic 38

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ar mandpositionedtodeterminethe(0,0)point.Theproperelectrodepositionwas thenrecalculatedbasedonthe(0,0)point.Theelectrodewasthenmovedoverthe exposedbraintissueandslowlyloweredtoadepthof2.8mm.Asmallamountofsuper gluewasusedtotemporallyholdeachelectrodeinplacebeforeapermanentgluewas applied.Afterthesupergluehadset,theelectrodewasreleasedfromthestereotaxic arm.Thisprocedurewasrepeatedforeachremainingbipolarelectrode.Finally,the referenceelectrodewasscrewedintoplace.Asmallamountofdentalcementwas mixedtotheconsistencythatwouldallowittoowaroundtheelectrodesandscrews. Thecavitycreatedbyretractingtheskin(Figure 3-1)waslledwiththedentalcement. Afterallowingthecementtocompletelycure,eachelectrodepinwasinsertedintothe pinholder(Figure 3-5).Thepincongurationwaskeptstandardamongstallanimals. Thedefaultsettingswas: 1.GroundPin 2.EmptyPin 3.RightCA1Pin-1 4.RightCA1Pin-2 5.LeftCA1Pin-1 6.LeftCA2Pin-2 7.EmptyPin 8.ReferencePin Oncetheelectrodeswereintheholder,askullcapwasbuiltuparoundtheholder tosecureitinplace.Thiswasdonebymixingdentalcementtoaputtylikeconsistency andapplyingitaroundthepinstrip.AdefaultbilateralCA1recordingheadstagecanbe seeninFigure 3-4. 39

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Micr owireimplantation Thegeneraltechniqueformicrowireimplantationdidnotvaryfromthatofany othersurgery.Theonlydifferencebetweenthesurgeryforabipolartwistandmicrowire surgery,wastherequirementofalargerwindowofthebraintobeexposed.Inorderto lowerthearrayarectangularsectionofskullhadtoberemoved.Basedonthedesired locationofthearray,thestereotaxicarmwasusedtomapoutthecenterandthenthe fourcornersofthearray(Figure 4-3). Usinga1/16 th inchballdrillbit,theoutlineofthearraywasmarkedontheskull accordingtoitscoordinates.Theoutlinewasrepeatedlytraceduntilthebonefragment becameloose.Thefragmentwasthenremovedwithtweezers.Thesmallsizeofthe microwireelectrodesmakesthemhighlysusceptibletodeectionduringtheimplantation processes.Inordertoreducethechancesofthetheelectrodesexing,thedurawas thenremovedfromthebrainwithasetofnetweezers.Thearraywastheloweredinto thebrainusingthestereotaxicarmasdescribedinSection 3.Atypicalcongurationcan beseeninFigure 4-3. EMGimplantation Formanystudiesitisimportanttodeterminethesleepstateoftheanimal.Thisis mostoftendonewithvideomonitoringandanalysisoftheanimal'selectromyography (EMG)trace.Beforebeginningthesurgerya2inchpieceof120micronwirewascut. 5mmofinsulationwasstrippedfromoneendand2mmofinsulationfromtheother end.AfterexposingtheskulloftheanimalasdescribedinSection 3,asmallincision (1cmlong)intheskinwasmadeabout2cmposteriortotheinitialincision.Thewire wasthreadedthroughan18gaugeneedleandthenthe5mmofexposewirewasfolded backontheneedleshafttocreateabarb(Figure 3-7 ).Theneedlewastheninserted intotheneckmuscle.Pressurewasappliedtothemuscleandtheneedlewasslowly removed. 40

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Ne xt,alargeboreneedlewassubcutaneouslyburrowedfromtheinitialincision tothesecondincision.Oncetheneedlewaspresentatthesecondincision,theEMG wirewasfeedtowardstheskull.AfterremovingtheneedletheEMGwirewasleftunder theskinsurface.Amalepinwascrimpedtotheexposedendofthewire.TheEEG electrodeswerethenimplantedasdescribedintheprevioussections.Followedbythe creationoftheheadstageasdescribedpreviously. PostSurgeryRecovery Followingallsurgeriesanimalsweremonitoredcontinuouslyuntiltheyregained theirrightingreexandmonitoredonceanhourfortherestoftheday.Immediatelypost surgery,Marcainewasdeliveredaroundthewoundtoprovidepainrelief.Afterremoval fromthestereotaxicframe,theanimalwasplacedinarecoverychamber,whichkeptthe animalwarmduringrecoveryviaatemperaturecontrolledheatingpad. StimulationProtocol Afterrecoveringfor1week,statusepilepticuswasinduced.Theanimalwas quicklyanesthetizedwithisourane(4%in1Lperminuteoxygen).Onceimmobile,a stimulatingcablethatconnectedtheanimaltoanA-MSystemscurrentstimulatorwas connectedtotherat.Theratwasallowedtocometofullconsciousinthestimulating chamberfor15minutes. Thedeliveredstimulusconsistedofa10secondtrainof1millisecond,50Hz biphasicsquarewavepulses.Each10secondtrainwasseparatedby2seconds.The stimulationamplitudeaveraged300microamps;however,thecurrentwastitratedonan animalbyanimalbasisifmorecurrentwasrequiredtoelicitaresponse. Duringthestimulationprotocol,theanimalwouldeliciteachgradeoftheRacine scale,culminatingingrade5seizures.Thephysicalmanifestationswouldbeginas wet-dogshakes,andafter30minutesconvulsiveseizureswouldbegin.Stimulation wouldcontinueuntilgradeveseizuresweredetectedevery5-10minutes(typicallyafter 60-90minutesofcontinualstimulation).Attheendofstimulation,afterdischargeswere 41

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detected forthenext12hours.Inadditiontothehyperexcitedelectrographicalactivity, seizureswouldcontinueoverthenextfourhours.Animalsthatneverachievedgrade5 seizuresordidnotmaintainanelectrographicalhyperexcitedstatehadalowchanceof developingspontaneousseizuresandwerethereforenotincludedinanystudies. AnimalHousing Atalltimestheanimalswerekeptinacontrolledenvironment.Temperatureand humiditywerekeptconstantandtheenvironmenthadaconsistent12-hrlight-darkcycle. Animalsweregivenfreeaccesstofoodandwateratalltimes. RecordingChambers Duringtimesofexperimentation,allanimalsweremovedintospeciallyconstructed housingthatwasdesignedtoaccommodatelongterm,continuousEEGandvideo monitoring.Whileanynumberofotherdesignswouldworkforlongtermhousing, severalkeycomponentsmustbeconsidered: HousingmustallowforthepassageofallcablesrequiredforrecordinganEEG signal Housingshouldbedesignedinsuchawaytominimizethelikelihoodofcatching thecableonthehousing. Housingshouldbecleartoallowforunobstructedviewingduringvideomonitoring Someelectronicsaresensitivetothebeddingmaterialgettingcaughtinfemale connectors.Therefore,thebestpracticeistodesignacagewhichdoesnotuse beddingandallowsalldroppingtofallthroughthebottomofthecage. AdesignthatmeetstheserequirementswasinitiallydescribedbyBertrametal. [6].Inthedesigna12inchtallx10inchwideclearacrylictubeisafxedtoastainless stealmeshwithopeningsbetween1/4and1/2inches.Siliconsealantwasusedtoafx themeshtothetube.Carewastakentomakethesealcompleteinordertoreduce thechancesoftheanimalbecomingstuckbetweenthetubeandwiring.Thewiring extended3inchespasttheperimeterofthetubeandthenwasbentdownwardonthe twoopposingsides;creatingaspaceforabeddingtraybelowthecage.A1/4holewas 42

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dr illedinthesideoftheacrylictube3inchesfromthebottomtoallowthepassageofa waterbottle'snozzleintothecage. EEGRecordings EEGrecordingsaretheprimarysourceofanalyzeddata.Unfortunately,once EEGrecordingshavebegunitistypicallyonlyamatteroftimebeforetheanimal's headstagebecomesdislodged.BecausetheSSSEmodelofepilepsytypicallydoes notproducespontaneousseizuresuntilfourweekspoststimulus,recordingsdidnot beginuntilfourweekspoststimulus.Doingsoprovidedthegreatestpossiblechanceof recordingspontaneousseizuresbeforetheanimalwasunusablebecauseofheadstage dislodgment. Fourweekspoststimulus,ananimalwouldbeinducedin4%isouranein1Lper minuteofoxygen.Oncetheanimalwasimmobile,itwasremovedfromtheinductionbox andaspeciallydesignedrecordingcablewasattachedtoitsheadstage.Theanimalwas thenplacedinarecordingchamberandconnectedtotherecordingsystem.Afterthe animalregaineditsrightingreex,EEGrecordingswouldbegin. EachrecordingsessionwouldbeginwithacalibrationoftheEEGrecordinguntil. Duringthistimeastandard50microamppeaktopeakvoltagewouldbepassedintothe recordinguntil.Thissignalwasrecordedbythesystemandthenusedtocalibratethe EEG.AftertheEEGwascalibrated,therecordingsystem'sinputwasswitchedtothe animal'sEEG.Theanimalwasthenlefttorecordfortheremainderoftheexperiment. 43

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Figure 3-1.Exposedskullpriortomarkingthecoordinatesforelectrodeplacement Figure 3-2.Stereotaxicframe 44

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Figure 3-3.PlacementofElectrodesrelativetoanatomicallandmarks Figure 3-4.Viewofheadstageimmediatelypostsurgery.The1designatestherstpin. 45

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Figure 3-5.Illustrationofhowheadstagelookswhenelectrodesarebeinginsertedinto theappropriateholdinglocations Figure 3-6.SchematicofthelocationofmicrowireelectrodesforrecordingfromtheCA1 andDG.Therightrecordingarrayoftenhadtoberotatedforplacementof thestimulatingelectrode. 46

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Figure 3-7.ForEMGimplantationsurgeries,anEMGelectrodewasthreadedthrougha sterileneedandthenabarbwascreatedtosecuretheelectrodeintoplace. 47

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CHAPTER 4 PLATFORMDEVELOPMENT RecordingSystems Therecordingofelectricalactivityofthebrainisnotanewconcept[ 33].Advances incomputationpoweranddigitalstoragespacehasallowedforEEGdatarecordingsto progressfromanaloguetracesrecordedonpapertohighlysampleddigitalEEGssaved onterabytesizedharddrives.Thishasprovidedtheinvestigatorwiththeabilitytostudy longtermchangesintheepilepticbrain.Asystemforthelongtermstudiesinvolving EEGactivityneedstocontainseveralkeycapabilities.Primaryamongsttheseare: Theabilitytohighlysampledatasimultaneouslyfrommultipleanimals. Theabilitytocapturetimelockedvideo. ThereareanumberofEEGrecordingsystemsonthemarket[ 6, 12],unfortunately mostofthesesystemswillnotfullymeettherequirementsoflongtermdataacquisition withoutsomemodication. Inthesestudies,threeprimaryrecordingsystemswereused.Forhighlysampled, microwirerecordingsaTucker-DavisTechnologies(TDT)RX5wasused.Thesystem wascapableofcollecting32channelsofdatasimultaneouslyfromtworats.Itcontained 5DSPchipscapableof16bitdatacollectionatamaximumsamplingrateof50kHz. Thesystem'stwomajordownfallswereitslackoftimelockedvideoandthefactthat itwasnotdesignedforlongtermstudies.Tosolvetheproblemoftimelockedvideo, aseparatecomputerwasusedtocapturedigitalvideo.Customsoftwarewasused tooutputatimestampviaaserialport,whichwasoverlaidontothevideostream withanonscreendisplaymodule.ThisallowedEEGstobemonitoredandthenthe correspondingvideocouldbefoundviathetimestamp.Theproblemofthesystemnot beingintendforlongtermstudieswasagainxedwithcustomsoftware.Initially,the TDT'srecordinganddatamanagementsystemwasusedandtheleswereconverted andstitchedtogetherinsoftwarewritteninMATLAB.EventuallyTDT'ssoftwarewas 48

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completely replacedwithcustomsoftwarethathandleddatamanagementandrecorded alldatain16bitbinaryles. Theothersystemsusedweremoreclinicalintheirbackgroundandwereusedto recordEEGsignalsthatwouldbemorelikelytobeseeninaclinicalsetting(e.g.1kHz samplingrate,macroscopicrecordings).TherstofthesesystemswastheStellate Harmoniepackage.Thissystemworkedbyrstamplifyingtheanimals'EEGwitha GrassTechnologiesanalogueamplier.Theoutputoftheamplierwasthenpassedto aNationalInstruments(NI)digitalacquisitioncard.Stellatesoftwareontherecording computerhandledthedigitizationdoneontheNIcard.TheStellatesystemwascapable ofrecordinghighlysampledEEGdatawithtimelockedvideo;however,comingfromthe clinicalworld,itwasintendedtoonlyworkwithonepatientatatime.Thisproblemwas solvedbyrecordingmultipleratsunderonepatient.Customsoftware(Figure 4-1)was thenusedtoreadtheStellateproprietaryformat,separateitintomultiplesubjectsand writeitasauniversallyreadableint16binaryformat. Cablesandcommutators .Throughexperienceitwaslearnedthatcablesand commutatorscanbethelargestsourceofnoiseintroducedintoanEEGrecording.For thispurposespecialcarewastakenwhenconstructingthecablesandcommutators requiredforrecordings.Thereareanumberofcommutatorsonthemarket[ 12].The preferredcommutatorduringtheseexperimentswasthe6channelDragonyslipring commutator.Thesecommutatorsprovidedverylowtorqueandinjectedverylittlenoise intotherecordingwhenrotating. TheDragonycommutatorscomewithlongleadswithDaleconnectorsfor inputintothesystem.Unfortunatelytheseleadsaretoolongandwillbedestroyed inexperimentsbytheanimals.Beforeexperimentationcouldbegintheseleadsandthe Daleconnectorwerereplacedwithashorterleadwhichcontainedaconnectormore suitabletoouranimals'headstage.Thepressttopofeachcommutatorwasremoved, andthewiresinsidethecommutatorweresnipped.Thetopwasthenheatedwithahot 49

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air gun.Thismadeitpossibletoremovetheepoxythatheldthewiringinplace.The commutatorwasthenrebuiltwithnewwiringandamorecompactconnector. ElectrodeConstructionandUse Experimentalquestionsshoulddictatewhichelectrodeisused,whatmetalitis constructedfrom,andwhereitisimplanted.Afterweighingallthepossibleoptions electrodescanbeassembledinhouseorpurchased.Allelectrodesusedinthese experimentswereconstructedinhouse. BipolarTwistElectrodes Bipolartwistelectrodes,whetherintendedforaratoramouse,followthesame generalmethodsforconstruction.Theonlydifferenceisthatamouseelectrodesis typicallymadefroma125micrometerdiameterTeoncoated,stainlessstealwire,while ratelectrodesuse330micrometerdiameterwire.Ineitherinstance,constructionbegan witha3inchpieceofwire.Withasharpknife,1.5mmofinsulationwasremovedfrom thetipofeachend.Thewirewasthenconnectedtoamalepinbyeithercrimpingor solderingthewireandpintogether.AUwasthencreatedwiththewirebybendingthe wireandplacingbothpinstogether.Ahemostatwasthenclampedtothebottomportion oftheU.Whileholdingthetwopinstightly,thehemostatwastwisteduntilthejunction ofthewireswas1/4inchfromthepins(Figure 4-2).Priortosurgerytheelectrodeswere cuttolength,typically5mmfromthejunction. Microelectrodes Intheinitialphaseofthesestudies,manyoftherecordingsdealtwithmicrowire arrayrecordings.Intheseexperiments,theTDTsystemwasused.Thestandard connectionforelectrodesusingthissystemisthe18channelOmneticsconnector (Figure 4-3). Beforebeginningconstructionofthearray,ajigwascreatedtoaidinkeeping standardspacingbetweentheelectrodes.Thiswasdonebyaligningthreealligatorclips 50

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in aPCBminivise.Eachclipwasseparatedbyabout1inch.Eachalligatorclipwas thenusedtoholdapieceof105micronmesh(Figure 4-4 ). Aftermakingthejig,thestandardsolderingpointsontheOmneticsconnectorwere rstcutinhalf.Theconnectorwaspositionedbelowthethreemeshscreenswitha third-hand.CarewasthentakentolevelthethreemeshscreensandtheOmnetics connector.Sixteen,2-inchgold-platedtungstenwireswerethencut.Approximately2 mmofinsulationwasremovedfromoneendofeachwire.Next,theexposedendof wirewascarefullylowereddownthroughthethreelevelsofmesh.Usingamicroscope, thewirewassolderedtothesolderpointontheconnector.Onceset,thenextwirewas loweredthroughthemeshandsoldered.Eachelectrodewasseparatedby4holesin themesh,creatingaelectrodespacingof420microns.Afterallthewiresweresoldered totheconnector,theywerecheckedforelectricalshorts.Ifnoshortswerefound,the bottompieceofmeshwastrimmedandthenloweredtowardstheconnector.Asmall amountofCranioplastwasusedtothensecurethewiresandconnectortogetherwhile alsoinsuringthesmallpinswouldnotexandcreateashort.OncetheCranioplastset, a3inchpieceofteon-coated330micronsteelwirewasconnectedtothegroundand referencepin. 51

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Figure 4-1.Customsoftwaretoextractandthenseparatemultiplesubject'sdataform theproprietaryStellatedataformat. Figure 4-2.Top:Groundandreferenceelectrode.Bottom:Bipolartwistelectrodebefore beingcuttolength. 52

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Figure 4-3.18ChannelOmneticsconnectorusedfortheconstructionofmicrowirearray electrodes Figure 4-4.Viseholdingthreealligatorclip.Eachclipisholdingasmallpieceofmesh. BelowthejiganOmneticsconnectorcanbeseen 53

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Figure 4-5.SetupforTDTsystem 54

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Figure 4-6.Stellatesetup 55

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CHAPTER 5 RELATIONSHIPBETWEENSPIKERATEANDSEIZURE Introduction Numerousstudieshaveclassiedepilepsyasadynamicdisease[ 50, 59].Itis believedthatstudyingthesechangesindynamicscouldgiveaninvestigatorenough insightintothesystemtoallowforclosedloopmodulationofthesystem.TheEEG producedbyanepilepticbraintransitionsthroughmanystates,suchasfromnormal toictalandbacktonormalthroughsomeformofhysteresis[ 45](Figure 5-1).The dynamicslikelyarisefromthedegradationofthethebrain'snaturalcontrolmechanisms. Itisthehopeofthisstudytodetermineameasurewhichcouldbeusedinthearticial recreationofthiscontrol. Inordertoactonthebrain,amarkerisneededtobeinvestigatedthatcouldbe easilymeasured,andthenmonitoredasfeedbackinputwasadministered.Todate determiningwhattocontrolintheEEGhasbeenasignicantproblem.TheEEG doesnotpresentitselfashavingasetpointwhichcaneasilybecompared,suchas canbeseenincontrolsystemstomaintainasettemperature.Inthisexample,the desiredtemperatureisthesetpointthesystemcancompareitselftoamakeactionable decisions.Unfortunately,whendealingwithEEG,thedecisionmakingprocessesisnot asblackandwhite. WhenanalyzinganEEGforbiomarkersofseizures,twoclassesofmarkercanbe investigated.TheEEGcanbereadasatimeseries,andnumerousmeasuresfrom signalanalysistheorycanbeappliedtothesignaltoextractavaluefortheEEGover time.Patternscanthenbeinferredinthesetimesignalsandpossiblerelationships toseizurescanbemade.Thesecondtypeofmarkersareelectrographicalevents. Thesecanincludetheoccurrenceofactionpotentials,populationspikes,highfrequency oscillationsorothereventsclinicalneurologistshavebeentrainedtodetect.Once 56

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detected theseeventscanbecounted,convertedtoarate,orcomparedinmanyother waystodeterminearelationshiptoseizures. Methods Male225-260gSprague-Dawleyrats(n = 43)werepreparedasdescribedin detailinsection 3.Briey,bilateral,bipolartwistelectrodeswereimplantedintothe leftandrightCA1(AP-4.3mm,ML 2mm,DV-2.8mm)andastimulatingelectrode wasimplantedintotheventralhippocampus(AP-5.3mm,ML 4.9mm,DV-5.0 mm)(Figure 5-2).Theanimalswereallowedtorecoverfor1week.Aftertherecovery periodtheanimalswereinductedintostatusepilepticus(section 3).Followinga4week recoveryperiod,continuousEEGrecordingsbegan.Datawasrecordedat1kHzwith timelockedvideo.Recordingscontinueduntiltheanimalremovedit'sheadstageor itwasapparenttheanimalwouldnotseize.Duringtherecordingperiod,datawas screenedweeklyforseizures.Usingthetimelockedvideo,eachdetectedseizurewas cataloguedandgradedbasedontheRacinescale. Followingthecompletionofrecordingphase,alldatawasprocessesforpopulation spikes.Theprocessbeganbybandpasslteringthedatafrom0.7-125Hz.Then anyeventthatexceededathresholdset5 deviationsabovethemeanoftheabsolute valueoftheEEGwasextractedfromthecontinuousdataset.Thethresholdvaluewas calculatedeveryhourtoaccommodateforchangesinthesignalthatoccurredovertime. Theuseof5 wasbasedonpreviousworkdonein-houseforspikedetection[ 81].When athresholdcrossingwasencountered,0.5secondsofdatabeforeand1.0secondsafter thecrossingwasextracted.Afterthedetectionofaspike,thedetectorwasputintoa refractoryperiodfor1.5seconds.Ifanotherspikeoccurredwithin1.5secondsofthe previousspikeitwasignored.Thisrefractoryperiodhelpedtoreducenoisefrombeing detectedasaspike. Toclusterthespikesandremoveartifactevents,awellknowclusteringalgorithm wasemployed[ 22].First,thepeakofeachwindowwasdetermined.Toaccomplish 57

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this ,thedataabovethethresholdcrossingwasapproximatedwithapolynomialt.The peakofthistwasthenusedasthepeakofthespike.Principlecomponentanalysis willbeusedtosortthespikes.Itiscrucialtohaveproperalignmentofthespikesandto subtractthewaveformsmean.Ifnot,theanalysismightrepresentthisvariancerather thantheintendedvariancebetweenwaveforms.Withoutusingthepeakasthezerotime point,theinitial1.5secondwaveformwastrimmedsuchthatthepeakofthewaveform occurred0.15secondsintoanew0.45second,trimmedwindow.Duetonaturalartifacts thatarecommoninlongtermrecordings,manyeventswillbechewingormovementthat canoftendominateanEEGsignal.Theselargeartifactscanoftencauseproblemslater inthesortingroutine.Theirremovalisdonewithak-meansoutlierfunction. Aftertheoutliershavebeenremovedthentheinitialclusteringstepcanbegin.In therstclusteringphase,thespikeswerehighlyoverclustered.Therstclusteringsize wasnormallytentimesthenumberofclustersthatwereexpectedtobeseen.Inmost instances,alltruespikeswereexpectedtoeventuallybeclusteredintotwogroups. Theinitialclustersareformedbydividingthespikesintotwogroups.Themeanofone halfistaken.Asecondmeanwasthenmadebytakingtherstmeanandaddinga smallamountofnoise.Thespikesarethenclassiedasbelongingtothemeanwhich itistheclosest.Asthespikesareassignedtothenearestmean,themeanisupdated withweightgiventothenumberofspikesassignedtothatmean.Themeanswere updatedthreetimesbeforeanotherbisectionwasmade.Thisprocessofmovingand recalculatingthemeansandbisectingthedata,wasrepeateduntilthedesirednumber ofclustersweregenerated.Aftertheinitialclustershavebeenset,theclusterswere thencombinedintolargergroups.Theaggregationoftheclusterscontinueduntil onlyclustersremainedthatcorrespondedtoaspecicspiketype.Thedecisionto combineclusterswasmadeifthetwoclustershadahighinterfaceenergy,ornumber ofwaveformsneartheborderthatseparatedtheclusters.Agraphicaloverviewofthis processisshowninFigure 5-3. 58

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Anal ysis Ofthe43animalsinthisstudy,16remainedinthestudytoyieldchronicrecordings. Nofatalitieswerecausedbysurgeryorthemodelinductionprocess.Mostanimalswere removedfromthestudyduetoadislodgedheadstagethatoccurredduringthefour weekrecoveryperiodaftermodelinduction.Ofthe16animals,8eventuallyproduced 765spontaneousseizures.Thespikedetectionandclusteringalgorithmwascomputed onthisdatabaseofseizures.Afterspikeclustersweregeneratedforeachanimal's les,spikefellintooneofthreeprimarycategories:anupwardspike,downwardspike ornoise.Theupwardinectingspikewasseeninallanimalsandinbothrecording channels.Thedownloadspikewasonlyseeninsomeanimals,andthereforeitwas notascloselyscrutinizedastheupwardspike.Foreachanimalanidealupwardspike clusterwasdeterminedbyvisualinspection.Astheanalysisprogressed,theclusters ineachlewerecomparedtotheidealspike.Ifthespikeclusterbeingviewedhada 90%correlationtotheidealclusterthentimepointsofit'sspikeswererecordedforlater rateanalysis.Thiswasdoneforeachspikeclusterineveryle.Theresultingsorted spikesfromtheEEGwerebinnedinto1minutesegmentsandcounted.Thecountin these1minutesegmentsiscalledthespikerate( R ).Basedonthethemethodsused here,itisimpossibletoclassifythespikesintotheirclinicalsubgroups.Forthisreason allpopulationspikesaredetectedandsortedandsimplyclassiedasspikes. Thepurposeofthisstudyistodetermineiftherearechangesinoccurrenceof spikesratepriortoseizure.Fundamentally,theanalysiswilltrytodetermineifspikes canbeusedtodistinguishbetweenthepreictalandinterictalstate.Thisdistinctionwill bemadebycomparingtheamplitudedistributionsofinterictalandpreictalspikerates. Throughthistechnique,increasesordecreasesinspikeratecanbecharacterized.In additiontospikeratecomparisons,comparisonswillalsobemadebetweenthetrends duringapreictalandinterictaltimeperiod. 59

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A seizureprecursorcouldoccuratanytimescale,andtherefore,numerous preictaltimeperiodsshouldbeanalyzed.Basedonliterature,thepreictaltime( s ) wasconstrainedtofourdurations: s = f5,30,60,120g min[14, 16, 37, 38, 62, 73]. Becausetherecouldpossiblybepostictaleffectsfromtheseizureonspikeoccurrence, the30minutesofdataafteraseizurewasnotanalyzed[ 61].Foreach s ,thespikerate wasbinnedintononoverlappingwindowsofequallengthto s .Ifaninterictal-preictal timeperiodwasnotatleast s +30 minuteslong,thenitwasdiscarded. Todeterminetheseparabilityoftheinterictalandpreictaltimeperiods,receiver operatingcharacteristics(ROC)wereemployed.Thisshowedthesensitivityand specicitybasedonvaryingthresholds.TheROCcurveisaplotofthesensitivity( S )vs thefalsepredictiverate( FPR ). FPR isalso1-specicity.WhenconductingtheROC analysis,thehypothesisthatspikerateincreasedduringthepreictaltimeperiodswas used. S = TruePositive T ruePositive + FalseNegative (5) FPR = FalsePositive F alsePositive + TrueNegative (5) Thespikerateineachdatasetwasthennormalizedfrom0to1toaccountfor variabilityandthustoallowforadirectcomparisonbetweenanimaldatasets.Itwasalso investigatedifspikeratecouldbeusedtomakeinferencesintothelocationandextentof damageinthebrain. Evaluations.Severaldifferentapproachesweretakentoevaluatethespikerate dataandtrytodistinguishbetweeninterictalandpreictaltimeperiods.Twoevaluation typeswereused,witheachevaluationshavingvariousschemes.Thersttypeof evaluationwasspikerateanalysis,whichhadthreeevaluationschemes.Intherst scheme,thedistributionof R forallthepreictalperiodsoflength s werecomparedtothe distributionof R fortheinterictaltimeperiods.ThiswillresultinasingleROCcurvefor 60

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each s Thenextschemeemployedathresholdtotrytodetermineadifferencebetween interictalandpreictaltimes.Herethe R foralltheinterictaltimeperiodsarecombined tocreateasingleinterictaldistributionof R .AROCcurveisthengeneratedforeach preictaltimeperiodbycomparingittotheconstantinterictaldistribution.Theareaunder thecurve(AUC),isthenrecordedforeachROCcurveasaperformanceparameter. Becausechangesmayhappeninthebrainthatcausethebaselineringratetochange, thethirdschemewilluseanadaptivethreshold.Inthisnalschemethedistributionof thepreictal R iscomparedtothedistributionoftheinterictal R immediatelyprecedingit. Again,theAUCvalueisthenrecordedasaperformanceparameter. Thenextevaluationtypeistocomparethechangeinringratebetweenthe interictalandpreictaltimeperiods.Thisevaluationtypewillhavetwoschemes.Inthe rst,thepercentchangeinthemeanringrateswillbecompared.Forthisanalysis,the preictalmean( PE )isthemeanof R overthepreictaltimeperiodoflength s .Thiswillbe comparedtothetotalinterictalmean( TIM ),whichisthemeanof R foreachcomplete interictaltimeperiodandtothepreviousinterictalmean( PIM ). PIM isthemeanof R overthewindowoflength s justpriortothepreictalperiod.Forthisanalysis,allwindows of s lengthwillbenon-overlapping.Thepercentchangesbetweenneighboringwindows willthenbecomparedtotheirrespectivecontrolsviaastudentt-testtodetermineifthey arestatisticallydifferent.Thecontrolforthe TIM groupwillbethepercentchangesseen betweenadjacentinterictaltimesegments.Forthe PIM group,theinterictaltimeperiods willbefurtherbrokenintowindowsofsize s andthenthepercentchangeinthemeanof eachofthesewindow's R willbecalculated. Thenextschemeforthisevaluationtypeistoanalyzethetrendseeninpreictal windows.Heretheslopesoftrendlinesforpreictaltimesegmentswillbecompared totheslopesoftrendlinesforinterictalsegments.Preictalwindowsoflength s willbe extractedandtheremaininginterictaldatawillbewindowedintosegmentsoflength s .A 61

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student' st-testwillthenbeconductedtodetermineifastaticaldifferenceexistsbetween thetrendsseenininterictalandpreictaltimeperiods. DiscussionandResults Whenexaminingtheabilityforspikeratetoyieldinformationaboutseizures,the rstevaluationconductedlookedatthedistributionsoftheamplitudeinthespikes perminutevstimeplots(Figure 5-4)fortheinterictalperiodsandpreictalperiodsof length s .Figure 5-5 showsthatthemeansofthesedistributionsweresignicantly different.Usingastudent'st-test,the5minutepreictaltimeperiodhadthehighest, butstillsignicant,pvalueof0.045.Allothervaluesfor s hadapvalue < 0.001.In eachcasethepreictaltimeshowedanincreasedringratewhencomparedtothe correspondinginterictaltime.ConductinganROCcurveanalysisonthesevaluesdid showthedistributionswerehighlyoverlapping.Foreachvalueof s ,theareaunderthe curveneverdeviatedfrom0.5bymorethan0.1.Thisindicatesthatwhilethegroupsare staticallydifferentfromoneanother,asimplethresholdingoperationisnotsufcientto separatethedistributions. Next,thedistributionofeachpreictalwindowwascomparedtothedistributionof allinterictaltimeperiodsandthedistributionoftheinterictaltimeperiodimmediately precedingthepreictalwindow.ThesedistributionswereusedtogenerateROCcurves. Thisyieldeda AUC andpvalueforcomparison.Table 5-1 showswhatpercentageof preictaldistributionswasstaticallydifferentthantheinterictaldistributions.Increasing thesizeof s alsoincreasedthelikelihoodofgeneratingapreictalwindowthatwas signicantlydifferentthantheinterictalperiod.Generatingaconstantthresholdby groupingallinterictaltimeperiodstogetheralsoshowedbetterresultsinevery s when comparedtotryingtodistinguishapreictaldistributionfromitsprecedinginterictal distribution. Seekingtoinvestigatetheimportanceindifferentspansintimeonspikeratepeaks andminimums,meanringrateswerealsocomparedoverthepreictalwindows.First 62

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in evaluatingthetrendsseenduringthepreictalwindowandimmediatelypreceding interictalwindowofthesamesize,theslopeofalineartthroughthewindow's spikeratevstimeplotwasrecorded.Plottingthemeanofthesevalues,nostatistical differencewasseenintheslopeofany s (Figure 5-7).Thesmallestpvaluewas0.59in the5minutepreictalwindow. Next,thepercentchangeseenbetweenmeanringratesofadjacentwindows wascompared.Herethechangebetweenthemeanoftheringrateofanentire precedinginterictaltimeperiodwascomparedwiththemeanoftheringrateforthe preictalwindow.Additionally,thechangeinthemeanbetweenthepreictalwindow andainterictalwindowalsooflength s immediatelyprecedingthepreictalwindow wasstudied.Thepercentchangeinthesevalueswascomparedtoacontrolthatwas generatedbymeasuringthepercentchangeinthemeanringrateoverwindowsfrom theinterictaltimeperiods.Essentially,allseizuredata,postictaldata,andpreictal datawasremovedfromtherecording.Thentheremainingsignal,whichnowonly containedinterictaldata,waswindowedintobinsofsize s andthepercentchangein themeanringratebetweenadjacentwindowswascalculated.Figure 5-6 showsthat foralmostall s therewasasignicantdifferencebetweenthepercentchangeseen whenenteringthepreictalwindowandthechangebetweentheinterictalwindows.The differencebetweentheaveragechangebetweenthepreictalwindowandentireinterictal windowandtheaveragechangebetweentheinterictalwindowsfor s =5 wastheonly relationshipwhosedifferenceswerenotsignicant(p = 0.12).Allothercomparisons hadpvalueslessthan0.01. Spikesareamarkerfordamage .Thestudycontainedanumberofanimalsthat wereelectrographicallystimulatedtoinduceseizure,butfailedtodevelopseizures. Whencomparingthesenonseizinganimalstoanimalsthatdiddevelopseizures(Figure 5-11 ),itwasdeterminedthatnonseizing,butinjuredbrain,hadasignicantlyhigher normalizedspikerateinthedamagedhemispherewhencomparedtotheseizing 63

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animal' sinterictaldataset(pvalue = 0.0161).Inahemispherecomparison,the hemispherewhichreceivedthemodelinducingstimulationhadsignicantlyaverage higherspikerates(Figure 5-12 ).Whencomparingthesegroups,allbutoneanimal experiencedapvalueoflessthan0.02. Conclusion Thisstudyrepresentstherstlongtermstudyconductedtoshowtherelationship betweeninterictalspikesandseizureonset.Thestudywasabletodrasticallyincrease thenumberofseizuresanalyzedbyimprovingonthetechniquesoflongtermrecordings. Thisstudyyieldedseveralresults.Therstisthereisanincreaseinspikerate duringthepreictaltimeperiodwhencomparedtotheinterictaltimeperiod.This wasseenacrossallvaluesof s ;however,theseparationbetweenthegroupswas thegreatestforthewindowsizeof30minutesandgreater.Asdeterminedbythe performanceparameter, AUC ,the120minutewindowshowedthegreatestseparation betweengroupswithan AUC of0.60.Thiswasonlyaminimalimprovementoverthe AUC of0.59seenwiththe60minutewindow.The AUC valuesfromtheROCcurves doindicatethatspikerateisapoorclassierbetweentheinterictalandpreictalperiods whenallpreictaltimeperiodsarecomparedtoallinterictaltimeperiods. Whencomparingeachpreictalandinterictalperiodsindividually,betterresultswere seen.Asteadyincreasewasseeninthepercentofpreictalperiodsthatwereableto bedistinguishedfrominterictalperiodsas s wasincreased.Thelowestpercentagesee, wasbarelyabovechance,at58.13%forthe5minutewindow,andpeakedat85.0%for the120minutewindow. Inasystemthatishighlyapplicabletoseizureprediction,whenthemeanof windowsweretaken,therewasastaticallysignicantchangeinthemeanofwindows whenenteringapreictaltimeperiod.Forwindowsgreaterthan30minutesinlength, thechangebetweenneighboringinterictalwindowswaslessthan30%;however,the changeinmeanringratewhenenteringapreictalwindowwasalwaysstatistically 64

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g reaterthanthisforwindowsizesof30minutesorgreater.Thisindicatesthatseizures maybepredictedbylookingforabnormallylargevariationsinmeanspikerateovertime. Whencomparingthemeannormalizedspikeinthehourpriortoaseizuretothat ofanimalsthatwerestimulatedbutneverseized,theanimalswithstimulationsbutno seizuresshowedahighernormalizedmeanspikerate(Figure 5-11 ).Analyzingaverage spikeratealsorevealedinformationaboutspikerateandpossiblepathology.Almostall animalsexperiencedagreaternumberofspikes,andhigherspikerate,inthestimulated hemisphere,regardlessiftheyseizedornot.Thesespikesaregeneratedinpathological tissue.Onlyoneanimalshowedahigherspikerateinitscontralateralhemisphere (Figure 5-12 ),andthiswaspresentintheanimalthatgeneratedthegreatestnumberof seizures.Whenallmeanringrateswerenormalizedanddividedintostimulatedand contralateralgroupings,therewasshowntobea25.5%reductioninaverageringrate betweenthetwohemispheres(Figure 5-13 p < 0.0001). Inconclusion,spikesprovideusefulinformationintothependingoccurrenceofa seizure.Thisstudyshowedtherewasasignicantchangeinspikeratebeforeaseizure, whencomparedtointerictalintervals.Thestudyalsoshowedthereisachangeinmean spikeratewhentransitioningintoapreictaltimeperiod.Thespikecountcouldalsobe usedtodetermineepilepsyseveritysinceanimalsexperiencedgreaterspikecounts intheirdamagedhemispheres,andconversely,animalswithelevatedspikecountsin bothhemispheresexperiencedthegreatestnumberofseizureswiththehighestgrade seizures. 65

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Figure 5-1.Dynamicstatesseeninepilepsy 66

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Figure 5-2.Electrodesplacementofbipolarelectrodesandstimulatingelectrodefor chronicrecording 67

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Figure 5-3.TheprocesstakentoextractspikesfromEEGdata.(A)Eventscrossinga setthresholdareextractedforanalysis.Theeventsmarkedwiththeclosed circlewilleventuallybecometheupwardsspikes,thesquaredeventswill becomedownwardspikesandtheeventmarkedwithastartwilleventually beclassiedasnoise.(B)Plotofalleventsdetectedafteraninitial clustering.(C)PCAplotofeventsaftereventscategorizedasoutliershave beenremoved.(D)Plotoftheaverageshapeofthetwogroupsofspikes thatweredetected. 68

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Figure 5-4.Figureshowingtheregionsofinterestforthespikes/minutevstimeplot.The verticalredbarsindicateseizures.The30minutepostictaltimeperiodis discardedfromanalysis.Thepreictalperiodhasalengthof s minutes,here showntobe60.Theinterictaltimeperiodisoflength (t n +1 t n ) (30+ s ), where t isthelocationintimeofaseizure. 69

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Figure 5-5.Meanringratesperminutefordifferentpreictalwindowsizes( s )compared totheirrespectiveinterictaltimes.Thegureshowstheaverage,normalized ringrateperminuteforeachpreictalwindowsize.Eachpreictalaverageis comparedtoitscorrespondinginterictalaverage.Theinterictaltimewas30 minutesafterthepreviousseizureto s minutesbeforethenextseizure.Each preictalmeanwassignicantlydifferentthanitsinterictalcounterpart. s =5 minhadthelargestpvalueof0.045,whileallotherpairshadpvaluesless than0.001.Inallcircumstances,thepreictalperiodexperiencedanincrease inringrate. 70

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T able5-1.Percentageofpreictaldistributionsthatwerestaticallydifferentthaninterictal distributions. s (min) Threshold Type P ercentageDifferentfromControl 5 constant 58.13 adaptiv e 53.92 30 constant 75.77 adaptiv e 71.81 60 constant 82.24 adaptiv e 75.0 120 constant 85.0 adaptiv e 77.57 71

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Figure 5-6.Plotinthepercentchangeofthemeanofneighboringwindows.Thepercent changebetweenthemeanintheringratewascalculatedbetweenthe entireinterictalperiodandthepreictalperiodandbetweenthelast s sized windowoftheinterictalperiodandthepreictalperiod.Foreachwindowsize of s ,thechangebetweenneighboringwindowsofsize s duringtheinterictal periodactedasthecontroltowhichthepercentchangewhenenteringthe preictaltimezonewascompared.Resultsaregroupedbasedonthesizeof s alongthexaxis.Thegureshowsthatthepercentchangeinmeanring rateduringthetransitionintothepreictalwindowwasstaticallygreaterthan thepercentchangeseenbetweeninterictalwindowsforallexcept s =5 min. 72

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Figure 5-7.Averageofslopesfromtrendlinesforspikerateoverawindowofsize s Whencomparingtheslopesseeninthepreictaltimeperiodtotheslope seeninthewindowofthesamelengthimmediatelyprecedingthepreictal windowwithastudent'st-test,nosignicantchangewasdetected. 73

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Figure 5-8.ROCcurvecalculatedfromthedistributionofinterictalandpreictalring rates.Forthisgure s =60 minutesisshown.TheAUCforthisROCcurve is0.59,withapvalue < 0.001.Thisindicatesthedistributionsare signicantlydifferentfromoneanother;however,thelowvaluefortheAUC indicatestherewouldbeasignicantnumberoffalsepositivesifthis schemewasusedtodetectseizures.Inthisexample,apreictalwindowwill haveagreaterspikingratethen59%ofinterictalwindows. 74

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Figure 5-9.Plotofspikesovera7.5dayperiod.Spikerepresentationremainedconstant duringrecordings.Eachplottedspikeinthisgureistheaverageofallthe upwardspikesfoundduringa4hourrecordingsession. Figure 5-10.Plotofspikescountedperminuteovertheentiretyofoneanimal's recording.Theyellowlinesdenoteseizures. 75

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Figure 5-11.Normalizedspikerateinanimalsthatwereelectrographicallystimulated. Theanimalswerethengroupedintoseizingandnonseizinganimals. Animalsthatwerestimulatedbutneverexperiencedaseizurehadhigher ringrateswhencomparedtoseizinganimals.Thegureshowsthereisa staticaldifferenceinthenormalizedringratewhencomparingdatafrom stimulatedbutnonseizinganimalstointerictaldataofseizinganimals. Figure 5-12.Comparisonofthemeanspikeratespikeduringnonseizuretimesofboth nonseizingbutinjuredanimalsandseizinganimals.Themodelwas inducedinthehemispheredenotedbytheshadedbar.R1-R6are hemispherepairsfrom6individualanimals.Thegureshowsinbothtypes ofanimals,thedamagedsideofthebraintypicallycontainedahigher numberofspikesperminute.Signicantlydifferentgroupsaredenotedwith alineabovethegrouping. 76

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Figure 5-13.Normalizedaverageringratecomparingthestimulatedandcontralateral hemispheres.Thegureshowsthereastaticallyhigheraverageringrate seeninthestimulatedhemisphere. 77

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CHAPTER 6 CONCLUSIONS Thisworkwasabletoconstructanumberofsystems,andsupportinginfrastructure fortherecordingandanalysisoflongterm,highlysampleddata.Indoingsoithas beenabletoaddknowledgetoaeachoftheeldsissoughttoimprove.Therstwas toidentifyabiomarkerthatcouldpotentiallybeusedforthecontrolandmodulationof seizures.Thisworkshowedthereisacorrelationbetweenspikesandseizures. UsingROCcurvesitwasshownthedistributionofspikerateamplitudeisstatically differentbetweeninterictalandpreictalperiods.Additionally,thepercentchangein meanspikerateoverwindowsgreaterthan30minutesishigherwhentransitioning intopreictaltimeperiodsascomparedtothetransitionfromoneinterictalwindowto another.Thisstudyhasalsoshownthatspikerateisalsocorrelatedtotissuedamage, byshowingthespikeratewashigherinthehemisphereofthebrainthatwasstimulated toinducethemodelofstatusepilepticus.Additionally,thisworkhascharacterizedthe patternsofseizureoccurrenceinthismodelofepilepsy. Thisinformationandinfrastructure,inturn,canbeusedtooptimizecurrent technologiesbyimprovingthecurrentstateoftheartinelectricallystimulatingthebrain forseizurecontrol.Thisstudycouldleadtothedevelopmentofaclosedloopseizure modulatorthatactsondetectedspikerate.Thedetectionofspikesisaminimally computationallyendeavorwhichcouldbeimplementedintoday'spacingtechnology. Additionally,thisinformationcouldbeusedtotriggeranymeansoftherapy(e.g.genetic, pharmacological,orthermal). FinalRemarks Inconclusion,thisworkpresentacompletesystemforthechronicrecordingof EEGinfreelymovinganimals.Theworkthendeterminedabiomarkerwhichwas proventohaveadirectrelationshipwithimpendingseizures.Usingmethodsputforth toclusterinterictalspikes,itwasdeterminedthatinterictalspikesincreasedinrateprior 78

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to animpendingseizure.Thisinformationcanbeusedtocreateaclosedloopseizure preventionsystembasedonthemodulationofinterictalspikes. 79

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APPENDIX A FURTHERCONSIDERATIONS ThepropersetupofarecordingstationisparamounttoproperEEGsignalcapture. Pastexperimentshaveoftenfocusedonshortterm,lowsampledrecordings.Inthepast decadethemostcostprohibitivebarriertolongterm,highlysampleddatarecordings wasthecostofhighcapacityharddrives.Duringtheinitialphaseofthisbodyofwork,a singlerat'smulti-monthrecordingwouldrequireapproximately$5000inharddrives.The rapiddropdecreaseinthepricetostoredatahasfacilitatedthedevelopmentoflong termrecordingstations. Duetotheinherentcostsquestionposedbymostresearch,recordingstations aredesignedtorecordforshorttermsessions.Toovercometheselimitations,custom softwarewasrequiredthatwascapableofaccessingthehardwareandsavingit's outputindependentlyofthesoftwareintendedtobeusedbythemanufactureof therecordingequipment.Theprimarybulkontherecordingsdoneforthiswork wasrecordedoneitheracompletelycustomrecordingsystememployingaNation InstrumentsdataacquisitionboardortheStellateHarmonie.Thesesystemswere bothcapableofrecordinglongtermdatasetsandstoringthemintopropitiatoryle formats.Timelockedvideowasacrucialnecessitythatwasrequiredforthegrading ofseizureseverity.Becausenotwocrystalsputouttheexactsamefrequency(and thereforenotwocomputerscanbeindependentlytimelockedwithoneanother),video recordinghastobedoneonthesamecomputerastheEEGrecordingitselfordone onasecondcomputerthatreceivestimeinformationfromtherst.Becauseofthe intensecomputationalrequirementstorecordvideoandEEGatthesametime,video monitoringwasoftendoneonasecondcomputer.Whenthiswasdone,atimestamp wasgeneratedbytherstcomputerandthenoverlaidontothevideobeforeitwas recordedandstoredbythesecondcomputer.Thisoverlaywasgeneratedbyaprogram runningontherstcomputerwhichoutputtimeinformationviaaserialport.Thetime 80

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stamp wasthenoverlaidonthevideofeedasASCIItextwithaDecadeEngineering videooverlaysystem. ReducingElectrographicalNoise ExperiencehasshownthemosttechnicallychallengingaspectofrecordingEEG activityisgettingitfromtheanimaltoadataserverwithoutintroducingartifact.There isaconsiderableamountofnoisethatwillbepresentinthesignalnomatterwhat precautionsaretaken.Thisincludesnoiseinducedbytheanimalchewing,scratching andmoving.Someinherentnoisecanbemitigatedbypropermontageselection; however,biologicaldataisalwaysnoisy.Conversely,externalnoisecanbecontrolled. Thisnoisecaninclude60Hznoisefrompowersources,vibrationnoiseandnoise inducedbycablemovement.Inordertoremove60Hznoise,allcomputersandother electricaldevicesshould,includingcellphones,shouldbekeptawayfromtheanimal. Ifpossible,recordingsshouldbedoneinaFaradaycage.Computersandotherother devicesthathavemovingpartssuchasfansshouldnotbestoredonthesametable theanimalswillreside.Theelectrodesensitivityiscapableofdetectingtheseslight vibrations.Whenpurchasingacommutator,highqualitycommutatorssuchasDragony, willnotintroducenoiseintothesignalwhenthecommutatorswivels.Lowerquality commutatorscanofteninducenoiseorskipwhentheyrotate. RecordingSetup Mostoftherecordingsystemsusedinthisstudywereintendedtobeusedwith humans.Figure A-1 givesanoverviewofhowcapturedsignalsprogressfromtheanimal totherecordingcomputer.Ananimalwiththreerecordingsites(RS)willhavethree recordingelectrodepinsextrudingfromitsheadstage,longwithagroundandreference electrode.Thesepinswillbeconnectedtoacommutator'sinputsviaacustombuilt cable.Thedatawillexitthecommutatorandinterfacewithastandard10-20electrode congurationbreakoutbox(Figure A-2)viaanothercustomcable.Atthebreakoutbox, multipleanimal'sEEGwillfeedintotherecordingsystem.Itisbestpracticetohaveeach 81

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r atfeedintoaseparatecolumnonthebreakoutbox.TheGrassamplierwillgenerate thedesiredmontagesfromthesignalsinputintothebreakoutboxassingleended signals.Forthesestudies,themontagesusedwereeachchannelwiththesignalfrom thereferencechannelsubtracted.TheoutputfromtheoftheGrassamplieristhefed intothechannelsontheStellate'sdataacquisitionboard. DataConversion Alldatarecordedwiththesesystemswassavedintoapropitiatoryleformat. Inordertoanalyzethedataitmustrstbeextractedandconvertedintoausable leformat.Additionally,thesesystemswereintendtobeusedwithasinglepatient. Therefore,whattherecordingsystemviewedasasinglepatientactuallycontainedthe recordedEEGfrom4animals.INT16binarywaschosenfortheleformatofchoice. Lackingtheprecisiontostorethedatainitsnaturalstate,eachdatapointwasmultiplied byalargescalingfactorinordertobringitintotherangeofINT16'sprecision.Doing sosavedhalftheharddrivespacewhencomparedtousingaFLOAT32binaryformat withnoscalingfactor.Toconvertthedata,acustomGUI(Figure A-3)wascreatedto convertandseparatetheanimalsintotheirownles.Uponselectingthelestobe convert.TheGUIwouldprompttheusertoassignchannelstoeachrat.Afterchannel assignmentwascompletethesoftwarewouldconvertthedatabyrstgatheringthetime stampinformationandsortingthelesintothecorrectchronologicalorder.Itwouldthen readthroughtheles'headerinformationtocheckwhichmontagethetherecordings weremade.Thiswasamethodoferrorcatchingtoensurethatanimalswerenotmoved betweenstationsduringrecordingsessions,andthereinchangingthechannelthey appearedwhenviewingtheStellatesystem.Afterconvertingallthedata,theprogram wouldalsogeneratealoglethatcontainedinformationsuchas:oldandnewle names,recordingstartdateforeachle,recordinglength,andsamplingfrequency. Itwasalsocapableofextractingaggedinformationifitwaspresentinthedatales. 82

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Flags couldbeusedwhenreviewingthedatawiththeStellatesoftwaretoindicate interestingeventssuchaslargeamountornoiseorseizures. AdditionalDevices Inadditiontocustomcablesandexperimentaltechniques,anumberofcustom toolswerecreatedduringtheseexperiments.Therstofthesewasforasideproject studyingplacecellactivityinrathippocampus.Forthisexperimentanautomatedfood pelletdisperser(Figure A-4)wasrequired.ThedevicewasmodelledinSolidWorks2010 andthenconstructedwitha3Dprinter.Thedevicewasdesignedwithareplaceable ywheelthatcouldbeexchangedbasedonwhatsizefoodpelletwasbeingusedfor theexperiment.Onceinplacetheywheelwasrotatedwithasteppermotor.The steppermotorwascontrolledwithaAtmega328microcontroller.Themicrocontroller alsocontrolledanLCDwheretheexperimentercouldseetheexperimentalsettings suchasexperimentlengthanddeliveryrateofthepellets.Whenpelletswerereleased fromthedevicetheywouldpassthroughaphotogate.Thegatewoulddetermineifa pelletwasreleased.Ifapelletfailedtoloadintotheywheelandthereforefailedtobe released,thesystemwouldcatchtheerrorandcontinuetoattempttoreleasepellets untilonewasreleased. AnimalMovementTracking .Onepossiblebiomarkerinvestigatedwashigh frequencyoscillations.Afterthestudiesbegan,itbecameapparentthatmanyof thehighfrequencyoscillationsthesystemwasdetectingwerepossiblymotion artifact.Becausethestudieswerebeingconductedoverlongperiodsoftime,arat trackingsystemwasdevelopedthatpossessedthecapabilitytoquantifytheanimal's movements. Afterreviewingthestateoftheartintrackingsystems[ 41, 65, 67],itwasdecided thesimplestmeanstodetectiftheanimalmovedwasthroughtheuseofcustom softwarewrittenaroundaninfraredcamera.Amoreaccuratesystemcouldhavebeen buildaroundaHalleffectormultiplecamerasystem;however,itwasdeemedthata 83

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single camerasystemwouldprovidetheneededresults.TheHalleffectsystemwas rejectedoverworriesofinducingartifactsintotheEEGsignalwiththeuseofhigh magneticelds,andthemulticamerasystemwasrejectedbasedonthefactmovement detectionwasrequiredbutnotpreciselocationdata. Themotiondetectionsystemworkedbyrstattachingasmallducialmarkerto theheadstageoftheanimal.Themarkerwaspositionedinsuchawaythatitwasnot hiddenastheanimalmovedaboutthecage.Theinfraredcamerawasthenpositionedin frontofthecagetocapturethemovementoftheanimalthroughoutitsenvironment.The customsoftwarethenreadinthedatafromtheinfraredcameraat30frame/sec.From thereceivedimages,thex,y,andsizeofthemarkerwascalculatedandthenthedata wastimelockedtotheEEGdataandsaved.TheresultingoutputcanbeseeninFigure A-5.Inthegurethegreenverticallinesrepresenthighfrequencyoscillationsthat werefoundinthedata.Asthegureshows,manofthemcorrespondtomotionartifact. 84

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Figure A-1.GrassConnections 85

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Figure A-2.10-20electrodecongurationbreakoutbox 86

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Figure A-3.Conversionsoftware 87

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Figure A-4.Automatedratfeederthroughthedevelopmentprocess.A)Initialsketch,B) 3DCADrenderingandC)naldevice(withywheelremoved) 88

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Figure A-5.10secondplotoftheareaofaducialmarkerasdeterminedbytracking software.Theareaofthemarker,alongwithitsxandypositionwereused todeterminewhenaratwasmoving. 89

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APPENDIX B ADDITIONALEXPERIMENTS MethodsforHybridModel Animals(n=5)inthisexperimentalhybridmodelwererstmadetohavespontaneous seizuresbyfollowingtheprotocolsetforthbyLothmanetal.anddescribedinsection 3. Afterspontaneousseizuresweredetected,atitrationwasbeguntodeterminethelowest doseofPTZthatwasrequiredtoinitiateaseizure.PTZwasdissolvedinsterilesaline andadministeredintraperitoneallyindosesof10,25,or50mg/kg.Eachconcentration wasadministeredonceadayforthreeconsecutivedays.If,duringthistime,aseizure neveroccurredwithinthehourimmediatelyfollowingtheinjectionofPTZthenext highestconcentrationofPTZwasusedforthreedayintervalsuntiladosagethatcaused seizureswasdiscovered. Inallveanimalsnoseizureswereseenwhenadministeringasubconvulsant dosage(Figure B-1)ofPTZ.Allanimalsreliablyexperiencedseizuresaftera50mg/kg doseofPTZ.ThesendingswereinlinewiththeresultsseenintheworkofHolmeset al.Inthatstudy,whentryingtoproduceachronicmodelofepilepsy,animalsthatwere repeatedlyexposedtolessthen50mg/kgofPTZneverproducedconsistentseizures [36]. Forthepurposeoftryingtodevelopahybridmodel,while50mg/kgisbelowthe dosageusedformanyacutemodels,thedoserequiredtoinitiateaseizurewashigher thenexpected.Additionally,itwasimpossibletotitratethedosageinordertogenerate grade4or5seizures.ThePTZquicklywentfromhavingnoeffecttocausingsevere, generalizedtonic-clonicseizures.Theseizuresweresevereenoughtocausetheonly seizureinducedmortalityseeninthisbodyofwork. 90

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Preliminar yResultsforFutureExperiments DirectStimulationtotheCA1 Toexplorethispossibility,severalpreliminaryexperimentswereconducted.Inthe rst,animalswerepreparedastheywereinsection 5.Theseanimalswereimplanted withbiploarelectrodesineachCA1;however,theywereneverinducedintostatus epilepticus.Aftertheywerefullyrecoveredfromtheimplantationsurgery,acute experimentswereconductedinwhichtheywererecordeddailyduringwhichtimea single,lowerfrequencystimulationwasdeliveredtotherightCA1for10seconds.The currentwasincreasedeachdayfrom10to25 Aby5 Asteps. Comparingthespectralanalysisofthesignalsdirectlybeforeandpriortothe stimulationinjection,therewaslittlechangeinpowerofthesignaluntilthestimulation reached25 A.The25 Astimulationdrasticallyincreasedthepowerofthesignal below10Hz,includingtheinjectionofsignalsthatcouldemulateinterictalspikes. Basedonthepriorknowledgethisseriesofstudieshasyielded,directstimulationof theCA1mayprovideapromisingavenueforthemodulationofseizurebymimickingthe occurrenceofinterictalspikes. SeizureInterventionWithDirectStimulation Furtheringthiswork,apreliminarystudywasconductedtodetermineifseizures couldbeabortedusingdirectstimulationtotheCA1.Theanimals(n=4)wereprepared asdetailedinsection 5.Afterrecoveryanimalswereinducedintostatusepilepticsand thenallowedtorecoveruntilspontaneousseizuresbegan.Inordertocreateondemand seizures,thehybridmodel(section 2)wasused. Briey,animalswereintraperitoneallyinjectedwithPTZatthedosethatwastitrated tobetheleastconcentrationcapableofgeneratingaseizure.AfterinjectionofPTZ,the animalwasthenimmediatelyconnectedtotherecordingandstimulatingequipment. TheEEGwascontinuallymonitoreduntiltherstappearanceofanelectrographical 91

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seizure .Atthistimethecontrolstimulationwasmanuallystarted.Thestimulationwasa 30seconds,120Hzbiphasicsquarewavewithanamplitudeof20or100 A. Theresultsofthispreliminarystudyshowedthestimulationwasunabletostop, reducetheseverity,ordurationofseizures.Theanimals'seizuresdidnotvary fromthoseofcontrolanimals,whichreceivedPTZbutnocontrolstimulation.While theresultsofthisexperimentwerecontrarytowhatwasexpectedtohappen,the resultswerenotunexpectedafterworkingwiththehybridmodelinvolvingPTZ.When creatingthemodel,itwasimpossibletotitratethedosagetocreatepartialseizures. Unfortunately,becauseofthealreadyhyperexcitedstateoftheanimal'sneurological conditionallseizuresbecamegeneralized.Itishypothesizedthatduetothegeneralized natureoftheseizurestheywouldbeimpossibletopreventbylocalstimulationjustprior tothestartofaseizure.Oncetheseizuresbecamegeneralized,theircontrolcould probablyonlybedonewithageneralizedtreatmentthatwouldspreadtotheentirebrain suchasanAEDorvagusnervestimulation.Thisexperimentdoesleadcredencetothe needforacontrolsystemthatworksbymodulatinganEEGbiomarkerratherthentrying tointervenedirectlyonaseizure.Itisbelievedthisexperimentalsoshowsitwould bemoreefcienttomodulatebrainactivityratherthentrytostopaseizureafterithas began. VNSExperimentalSetup Vagusnervestimulationisapromisingnewtherapyforepilepsy.Severalanimals weretestedtoseeifitwaspossibletomimicavagusnervestimulationsetupwith equipmentalreadyavailableinthelab.Forsuchasetup,themostdifcultitemtomake isanelectrodetoconnecttothevagusnerve.Inhumanvagusnervestimulatorsthe developmentofanacceptableelectrodehasbeenoneofthemoststudiedaspectsof vagusnervestimulationresearch. AfterpreparinganimalswithelectrodesintheeachCA1,andadditionalelectrode wasalsoplacedintheabdomentodetectheartrate.Aftertheplacementofthese 92

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electrodes ,thevagusnervewasdissected.Anelectrodecontainingabarbedanode andcathodewascreatedtoallowforthepassageofcurrentintothevagusnever.The electrodewasattachedtothevagusnerveandthentunneledtothebackoftheskull whereitwasinsertedintotheanimalsheadstage.Becausethevagusnerveinnervates theheart,itcanbeusedtodetermineifcurrentisbeingdeliveredtothevagusnerve. Aftertheanimalrecoveredfromtheimplantationsurgeryforoneweek,theanimal wasconnectedtothestimulatorandrecordingsystem.TheanimalsheartrateandEEG wererecordedwhiledifferentamountsofcurrentweredeliveredtothevagusnerve. Unfortunately,theelectrodedesignfailedandtheheartratewasunchangedthroughout theexperiment(Figure B-2). Vagusnervestimulationisaninterestingareaoffuturestudy,unfortunatelywewere unabletomakeinhouseelectrodescapableofdeliveringcurrenttothevagusnerve. Thisisnotanendpointforthestudyofvagusnervestimulationinthislab,asthereare manufacturesofelectrodesthathavebeenshowntoworkontheratvagusnerve. 93

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Figure B-1.Averagenumberofseizuresseenwhenadministering10,25,or50mg/kgof PTZ.Thegureshowsthatnoseizureswereproducedwith10or25mg/kg ofPTZ.Theonlyconcentrationthatproducedanyseizures,50mg/kg,is reportedtodothesameinnormalanimals.Thisprovesthehybridmodelis notviable. 94

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Figure B-2.Heartheartbeforeandduringthedeliveryofstimulationthroughthevagus nerve.Nochangeisseenintheheartrate.Itcanthereforebeassumedthat nocurrentisbeingdeliveredtothevagusnerve. 95

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REFERENCES [1]A voli,M.Dointerictaldischargespromoteorcontrolseizures?Experimental evidencefromaninvitromodelofepileptiformdischarge. Epilepsia (2001). [2]Avoli,M,Biagini,G,anddeCurtis,M.Dointerictalspikessustainseizuresand epileptogenesis? EpilepsyCurrents (2006). [3]Avoli,M,D'Antuono,M,Louvel,J,Kohling,R,Biagini,G,Pumain,R,D'Arcongelo, G,andTancredi,V.Networkandpharmacologicalmechanismsleadingto epileptiformsynchronizationinthelimbicsysteminvitro. ProgressinNeurobiology 68(2002).3:167. [4]Barbarosie,MandAvoli,M.CA3-Drivenhippocampal-entrohinalloopcontrols ratherthansustainsinvitrolimbicseizures. JournalofNeuroscience 17(1997): 9308. [5]Bertram,E.Functionalanatomyofspontaneousseizuresinaratmodeloflimbic epilepsy. Epilepsia 38(1997).1:95. [6]Bertram,EH,Williamson,JM,Cornett,JF,Spradlin,S,andChen,ZF.Designand constructionofalong-termcontinuousvideo-EEGmonitoringunitforsimultaneous recordingofmultiplesmallanimals. BrainResearchProtocols 2(1997).1:85. [7]Brenner,R.EEGinconvulsiveandnonconvulsivestatusepilepticus. Journalof ClinicalNeurophysiology 21(2004):319. [8]Brown,TandHolmes,G.Epilepsy. NewEnglandJournalofMedicine 344 (2001).15:1145. [9]Buzsaki,G,Leung,LS,andVanderwolf,C.H.CellularbasesofhippocampalEEG inthebehavingrat. BrainResearch 6(1983):139. [10]deBittencourt,P,Adamolekum,B,Bharucha,N,Carpio,A,Cossio,O,Danesi,M, Humas,M,Meinardi,H,Ordinario,A,Senanayake,N,Shakir,R,andSotelo,J. EpilepsyintheTropics:I.Epidemiology,socioeconomicriskfactorsandetiology. Epilepsia 37(1996).11:1121. [11]deCurtis,MandAvanzini,G.Interictalspikesinfocalepileptogenesis. Progress inneurobiology 63(2001).5:541. [12]delCampo...,C.EEGrecordinginrodents,withafocusonepilepsy. ...in neuroscience/editorialboard (2009). [13]Dreifuss,F.Classicationofepilepticseizures. Epilepsy:ACompreshensive Textbook.Lippincott-RavenPublishers,1997. 96

PAGE 97

[14]Duc krow,RBandSpencer,SS.Regionalcoherenceandthetransferofictal activityduringseizureonsetinthemedialtemporallobe. Electroencephalography andClinicalNeurophysiology 82(1992).415-422. [15]Eileen,P,Freeman,J,andetal.Amulticenterstudyoftheefcacyoftheketogenic diet. ArchieveofNeurology 55(1998).11:1433. [16]Elger,CEandLehnertz,K.Seizurepredictionbynon-lineartimeseriesanalysisof brainelectricalactivity. EuropeanJournalofNeuroscience 10(1998).786-789. [17]Engel,J. Surgicaltreatmentoftheepilepsies .Raven,1987. [18]Engel,JandAckermann,R.InterictalEEGspikescorrelatewithdecreased,rather thanincreased,epileptogenicityinamygdaloidkindledrats. BrainResearch 190 (1980):543. [19]Engel,JandSchwartzkroin,P.Whatshouldbemodeled? ModelsofSeizuresand Epilepsy.ElsevierAcademicPress,2006. [20]Engel,JandShewmon,D.Overview:whoshouldbeconsideredasurgical candidate? Surgicaltreatmentoftheepilepsies .Raven,1993. [21]Engel,JeromeandPedley,Timothy. Epilepsy:ACompreshensiveTextbook ,vol.1. Lippincott-RavenPublishers,1997. [22]Fee,M,Mitra,P,andKleinfeld,D.Automaticsortingofmultipleunitneuronal signalsinthepresenceofanisotropicandnon-Gaussianvariability. Journalof NeuroscienceMethods 69(1996):175. [23]Fisher,RobertandSaul,Maslah. OverviewofEpilepsy .2010. [24]forDiseaseControl,CenterandPrevention.Oneofthenation'smostcommon disablingneurologicalconditions.2007. [25]Freeman,J,Eileen,P,andetal.Theefcacyoftheketogenicdiet-1998:A prospectiveevaluationofinterventionin150children. Pediatrics 102(1998).6: 1358. [26]George,R,Salinsky,M,andKuzniechy,R.Vagusnervestimulationfortreatmentof partialseizures.Long-termfollow-uponrst67patientsexistingacontrolledstudy. Epilepsia 35(1994):637. [27]Geyer,JandCarney,P.Seizuresemiology:thecharacteristicsoftheseizure. ReadingEEGs .LippincottWilliamsandWilkins,2010. [28]Gloor,P.Neurobiologicalsubstratesofictalbehavioralchanges. Advancesin Neurology 55(1991):1. [29]Goddard,G.Developmentofepilepticseizuresthroughbrainstimulationatlow intensity. Nature 214(1967):1020. 97

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[30]Gotman, J.Relationshipsbetweentriggeredseizures,spontaneousseizures, andinterictalspikinginthekindledmodelofepilepsy. ExperimentalNeurology 84 (1984):259. [31]Gotman,JandMarciani,M.Electroencephalographicspikingactivity,druglevels ansseizureoccurrencesinepilepticpatients. AnnNeurology 17(1985).597-603. [32]Gotman,JM.Relationshipsbetweeninterictalspikingansseizures:humanand experimentalevidence. CanadianJournalofNeurologicalScience 18(1991): 573. [33]Greeneld,L,Geyer,James,andCarney,Paul. ReadingEEGs .LippincottWilliams andWilkins,2010. [34]Harbord,M,Grattan-Smith,J,andDesmond,P.Temporallobeepilepsyinchildren: reappraisalofetiologyandoutcome. PediatricNeurology 3(1987):263. [35]Hauser,WA,Annegers,JF,andRocca,WA.Descriptiveepidemiologyofepilepsy: contributionsofpopulation-basedstudiesfromRochester,Minnesota. MAYO CLINICPROCEEDINGS 71(1996).6:576. [36]Holmes,G,Sarkisian,M,andBen-Ari,Y.Mossybersproutingafterrecurrent seizuresduringearlydevelopmentinrats. JournalofComputationalNeurology 404(1999):537. [37]Iasemidis,LD,Pardalos,P,andSackellares,JC.Quadraticbinaryprogramming anddynamicalsystemapproachtodeterminethepredictabilityofepileptic seizures. JournalofCombinatorialOptimization 5(2001).9-26. [38]Iasemidis,LDandSackellares,JC. Measuringchaosinthehumanbrain ,chap.The evolutionwithtimeofthespatialdistributionofthelargestLyapunovexponenton thehumanepilepticcortex.WorldScientic,1991,49. [39]Janszky,J,Fogarasi,A,Jokeit,H,Schulz,R,Hoppe,M,andEbner,A. Spatiotemporalrelationshipbetweenseizureactivityandinterictalspikesin temporallobeepilepsy. EpilepsyResearch 47(2001).3:179. [40]Jensen,M,Azouz,R,andYaari,Y.Variantringpatternsinrathippocampal pyramidalcellsmodulatedbyextracellularpotassium. JournalofNeurophysiology 71(1994):831. [41]Knutsen,P,Derdikman,D,andAhissar,E.Trackingwhiskerandheadmovements inunrestrainedbehavingrodents. JournalofNeurophysiology 93(2005): 2294. [42]Kotagal,P,Rothner,A,Erenberg,G,Cruse,R,andWyllie,E.Complexpartial seizuresofchildhoodonset:aveyearfollowup. ArchieveofNeurology 44 (1987):1177. 98

PAGE 99

[43]K upferberg,H.Animalmodelsusedinthescreenginofantiepilepticdrugs. Epilepsia 42(2001).Suppl4:7. [44]Lange,H,Lieb,J,Engel,J,andCrandall,P.Temporo-spatialpatternsofpre-ictal spikeactivityinhumantemporallobeepilepsy. Electroencephalographyand ClinicalNeurophysiology 56(1983):543. [45]LDIasemidis,RSSavit,JCSackellares. NonlinearDynamicalAnalysisoftheEEG chap.QuanticationofhiddentimedependenciesintheEEGwithintheframework ofnonlineardynamics.WorldScientic,1993,30. [46]Lindsay,J,Ounsted,C,andRichards,P.Longtermoutcomeinchildrenwith temporallobeseizures.IV:Geneticfactors,febrileconvulsionandremissionof seizures. DevelopmentalMedicineandChildNeurology 22(1980):429. [47]Litt,BandEchauz,J.Predictionofepilepticseizures. TheLancetNeurology 1 (2002).1:22. [48]Lothman,E,Bertram,E,Bekenstein,J,andPerlin,J.Self-sistaininglimbicstatus epilepticusinducedbycontinuoushippocampalstimulation:electrographicand behavioralcharacteristics. EpilepsyResearch (1989).107-119. [49]MacDonald,R.CellularEffectsofantiepilepticdrugs. Epilepsy:ACompreshensiveTextbook .LippincottWilliamsandWilkins,1997. [50]Mackey,MCandGlass,L.Oscillationsandchaosinphysiologicalcontrolsystems. Science 197(1977):287. [51]Mangan,PandBertram,E.Shortened-durationGABAAreceptor-mediated synapticpotentialsunderlieenhancedCA1excitabilityinachronicmodelof temporallobeepilepsy. Neuroscience 80(1997).4:1101. [52]Mangan,PandLothman,E.Profounddisturbancesofpre-andpostsynaptic GABAB-receptor-mediatedprocessesinregionCA1inachronicmodeloftemporal lobeepilepsy. JournalofNeurophysiology 76(1996):1282. [53]Marsh,E,Peltzer,B,Brown,M,Wustoff,C,Storm,P,Litt,B,andPorter,B. Interictaleegspikesidentifytheregionofelectrographicseizureonsetinsome,but notall,pediatricepilepsypatients. Epilepsia 51(2010).4:592. [54]Mathern,G,Babb,T,andArmstrong,D.Hippocampalsclerosis. Epilepsy:A CompreshensiveTextbook .Lippincott-RavenPublishers,1997. [55]Mathern,G,Babb,T,andetal,JLeite.Thepathogenicandprogressivefeatures ofchronichumanhippocampalepilepsy. EpilepsyResearch 26(1996):151. [56]Mazarati,A,Thompson,K,andetal,LSuchomelova.Statusepilepticus:electrical stimulationmodels. ModelsofSeizuresandEpilepsy .ElsevierAcademicPress, 2006. 99

PAGE 100

[57]McIntyre ,D.ThekindlingPhenomenon. ModelsofSeizuresandEpilepsy ElsevierAcademicPress,2006. [58]McLachlan,R.Vagusnervestimulationforintractableepilepsy:areview. Journal ofClinicalNeurophysiology 14(1997).5:358. [59]Milton,JG. Selforganizedbiologicaldynamicsandnonlinearcontrol ,chap. Epilepsy:multistabilityinadynamicdisease.CambridgeUniversityPress,2000, 374. [60]Morest,DK.Experimentalstudyoftheprojectionsofthenucleusofthetractus solitariusandtheareapostremainthecat. JournalofComputationalNeurology 130(1967):227. [61]Mormann,F,Kreuz,T,Rieke,C,Andrzejak,R,Kraskov,A,David,P,Elger,C,and Lehnertz,Klaus.Onthepredictabilityofepilepticseizures. ClinicalNeurophysiology 116(2005).569-587. [62]Navarro,V,Martinerie,J,Quyen,MLeVan,Clemenceau,S,Adam,C,and Baulac,M.Seizureanticipationinhumanneocorticalpartialepilepsy. Brain 125(2002).640-655. [63]Nehlig,AanddeVasconcelos,P.Themodelofpentylenetetrazol-inducedstatus epilepticusintheimmaturerat:shortandlongtermresults. EpilepsyResearch 26 (1996):93. [64]Nistri,AandConstanti,A.Pharmacologicalcharacterizationsofdifferenttypes ofGABAandglutamatereceptorsinvertebratesandinvertebrates. Progressin Neurobiology 13(1979):117. [65]Noldus,L,Spink,A,andTegelenbosch,R.EthoVision:aversatilevideotracking systemforautomationofbehavioralexperiments. BehaviorResearchMethods, InstrumentsandComputers 33(2001).3:398. [66]Olsen,R.TheGABApostsynapticmembranereceptor-ionophorecomplex. Siteofactionofconvulsantandanticonvulsantdrugs. MolecularandCellular Biochemistry 39(1981):261. [67]Parperno,E,Sasada,I,andLeonovich,E.Anewmethodformagneticposition andorientationtracking. IEEETransactionsonMagnetics 37(2001).4:1938. [68]Paxinos,GandWatson,C. Theratbraininstereotaxiccorrdinates .Academic Press,1998. [69]Penry,JandDean,J.Preventionofintractablepartialseizuresbyintermittent vagalstimulationinhumans:preliminaryresults. Epilepsia 31(1990).S2:40. [70]Peterson,SandAlbertson,T. Neuropharmacologymethodsinepilepsyresearch CRCPress,1998. 100

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[71]Pitkanen, A,Schwartzkroin,P,andMoshe,S. ModelsofSeizuresandEpilepsy ElsevierAcademicPress,2006. [72]Porter,RandMeldrum,B.Overview:Antiepilepticdrugs. Epilepsy:ACompreshensiveTextbook .LippincottWilliamsandWilkins,1997. [73]Quyen,MLeVan,Martinerie,J,Baulac,M,andVarela,F.Anticipatingepileptic seizureinrealtimebyanonlinearanalysisofsimilaritybetweenEEGrecordings. NeuroReport 10(1999).2149-2155. [74]Racine,R.Modicationofseizureactivitybyelectricalstimulation:II.Motor seizure. ElectroencephalographyandClinicalNeurophysiology 32(1972): 281. [75]Renfroe,JBandWheless,JW.Earlieruseofadjunctivevagusnervestimulation therapyforrefractoryepilepsy. Neurology 59(2002).90064:26. [76]Ricardo,JAandKoh,ET.Anatomicalevidenceofdirectprojectionsfromthe nucleusofthesolitarytracttothehypothalamus,amygdalaandotherforebrain structuresintherat. BrainResearch 153(1978):1. [77]Rutecki,P.Anatomical,physiological,andtheoreticalbasisfortheantiepileptic effectofvagusnervestimulation. Epilepsia 31(1990).S2:1. [78]Shin,CandMcNamara,J.Mechanismofepilepsy. AnnuRevMed (1994). [79]Slutzky,MW,Cvitanovic,P,andMogul,DJ.Manipulatingepileptiformbursting intherathippocampususingchaoscontrolandadaptivetechniques. IEEE TransactionsonBiomedicalEngineering 50(2003).5:559. [80]Staley,KJandDudek,FE.Interictalspikesandepileptogenesis. Epilepsy Currents 6(2006).6:199. [81]Talathi,SachinS,Hwang,Dong-Uk,Ditto,WilliamL,Mareci,Tom,Sepulveda, Hector,Spano,Mark,andCarney,PaulR.Circadiancontrolofneuralexcitabilityin ananimalmodeloftemporallobeepilepsy. NeuroscienceLetters (2009):1. [82]Tatum,W,Vale,F,andAnthony,K. Apracticalapproachtoneurophysiological intraoperativemonitoring ,chap.EpilepsySurgery.2008. [83]Ulbert,I,Halgren,E,Heit,G,andKarmos,G.Multiplemicroelectrode-recording systemforhumanintracorticalapplications. JournalofNeuroscienceMethods 106 (2001).1:69. [84]Wiebe,S,Blume,W,Girvin,J,andEliasziw,M.Arandomized,controlledtrialof surgeryfortemporallobeepilepsy. NewEnglandJournalofMedicine 345(2001): 311. [85]Wieser,H.PreictalEEGndings. Epilepsia .vol.30.1986,664. 101

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[86]Wieser ,HG.Mesialtemporallobeepilepsywithhippocampalsclerosis:Reportof thecommissiononneurosurgery. Epilepsia 45(2004):695. 102

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BIOGRAPHICAL SKETCH StephenMatthewMyerswasborninPanamaCity,Florida.Stephenisthesonof SteveandMaryJoMyersandhasoneyoungerbrother,Jordan.Aftergraduatingfrom BayHighSchoolin2001,StephenattendedGeorgiaTech.Stephengraduatedwithhigh honorsfromGeorigaTechwithaB.S.inbiomedicalengineering. StephenwasawardedanAlumiFellowshiptopursueaPh.D.inbiomedical engineeringattheUniversityofFlorida.WhileworkingonhisPh.D.,Stephenearned hisM.E.inbiomedicalengineeringfromtheUniversityofFlorida.Stephen'sresearch interestinvolveneuroengineering,epilepsy,humanfactorsengineeringandusability. StephenhasbeenmarriedfortwoyearstoRenee,andtheycurrentlyhavetwo dogs,CooperandTori. 103