CEREBRAL ISCHEMIA, SPATIAL MEMORY AND LOCOMOTOR

CEREBRAL ISCHEMIA, SPATI AL MEMORY AND LOCOMOTOR ACTIVITY

by

©DongWang M.B.

A thesissubmittedtothe Schoolof GraduateStudies in partialfulfillmentoftherequirements for the degreeof

MasterofScience

Divisionof Beslc MedicalSciences Faculty of Medicine MemorialUniversityof Newfoundland

September6 th,1990

"

~\

St.John's Newfoundland

1"'1

The author has granted anitrevocablenon- .xcluslvellcenceeJlowIngtheNaIionaIUbmy ofcanadatoreproduce,loan,cflSlributeorsell copies of hislher thesisbyeny meansandIn eny formor format,maIdogthisthesisavailable

to interestedpersons.' .

The author retainsownershipofthecopyl1ght in his/herthesis.':Neitherthethesisnor substantialextracts fromitmaybeprintedor otherwisereproduced without hislherper- mission.

l'suteur aaccordeunelicenceil'l'\Wocab&eet nonexclusivepennettanti\ laBi~ue nationaleeuCanadadereprodulre, pritter.

oLStribuer au vendredescopesdesa tMse de quelquemaniereet sousqueIqueforme que cesoit pour mettredesexempWresde cette these

a

Iadisposition des persoones jnteress ees .

L'auteur 0005e(Vetapropnetedudroitd'atJteur quiprotegesathese.Nill!tMseoldesextralts substantielsde cene-ct ne doivent litre lmpcimes au autrarnentreproduits sans son autorisation .

ISBN0-315-65320- 5

To

My Mother, Father and Sisters.

ACKNOWLEDGEMENTS

Iwouldlike toexpressmy thanks10Dr.D.Corbellforprovidingme with the opportunityto work on thisprojectand for his encouragement.guidanceand supportthrough all stages of thisresearch.Iwouldalsoliketo thankDr.R.

Neumanfor hiswisecouncil and encouragement,Dr.P.Moody-Corbettfor her supportand helpfulcomments,andMs.S.Evansfor hertechnical support.

Iamgratefulfor the financialsupportprovided bythefellowshipfrom the SchoolofGraduateStudiesand thebursary from the Faculty 01Medicine.

withoutthissupport11would not havebeen possiblefor me to study here.

iv

ABSTRACT

Strokeisamong oneof the leadingneurologicaldisordersinclinics.The purposeofthisstudy wasto investigatethe functionaldeficits ofthe experimental -stroke- animal.In orderto gel a betterunderstanding of thereal mechanismsbehindthesedeficits,a systemallcobservation of the behavioural and pathologicalchangesassociatedwith cerebralischemiawas carriedout i,l threeexperimentsInMongoliangerbils.

InEKperimenl 1, an animalmodel using delayed,repetinve cerebralIsche~ la was usedtodeterminethebehavioural andneuropathological changes following multi-episodes01cerebralIschemia.

InExperiment2,animals werepre-exposedtothe lest environment before ischemiaandan attemptWdSmade to determinewhetherthepost-ischemic hyperactivity reSUlted from a simplechangein motorfunction oradefICitin spatialmapping ability.

In Experiment 3,the M8l1'opathOlogicalandlocomotoractivitychanges resulting fromdifferentcarotidarteryocclusionduratiort:l(5,10and15minutes) wereinvestigated.Anattemptwas madetodeterminewhether thegraded ischemiaresultingfromthedifferent ischemicdurationsresulted ingraded increasesInlocomotoraC1lvity.

From thesethreeexperimentsitisconcluded thaIthe basisfor theincreased locomotor activityfollowInganepisodeof cerebralischemia isanalteration of spatiallearningabllily.

TABLE OF CONTENTS

ACKNOWLEOG EM ENT ---....---. -.-••--••---.---...-.-. III ABSTRACT••-••- -••••---••••••••--- --.---.--Iv TABLE OF CONTENTS..---v LIST OF FIGURES --- ---... ---.--- --- - --- vII LIST OF TABLES---••••••---vIII LIST OF ABBREVIATIONS ---•••••••---.---.-••Ix

GENERAL INTRODUCTION--- --- --- ----1 1.Cerebral ischem ia ---••••----.-•••••- --.--•••---••---...1 2.Neuroanatomyof hippocampus.-- -••----••••.•--.-- --.--•••5 3.Excitatory am inoacids .--.- ---- - ••-••••••••- - - 6 4.Functional studi es••••••••-•••••••-_._-_._ 12

EXPERIMENT ONE --- .-••••••--- -- --- 16

Introduction -•••••••••••••••••__._ ••__ _.. - 17 Methods- ••- - - - --•••- --.-.- ••-- -- --.17 Hesults••-- -••-.- - - _-.-•••---_ .._- ----•••-- -- --. 25 Discussion - - - -- -.---.-••---- •••---. -.---- - ••••- -•••• 28

EXPERIMENT TWO.-...-•••••--- -- --- ----.---35

vi

Introduction-_.._-_.••.•.-•••-••••••••.__._.••..•_._••__..••-_.•_--·36 Methods-- --....•.•...- ..•.-----.-.--- ---.-.---.•..._-_••••-36 Results--.--.--.---...•.•.----.•..--.-.•..-.--- •.••.••••--.••.---- 39 Discussion-- ..-..---...•.--.---.•...•.-- -.••- ---._-47

EXPERIMENTTHREE•••••...•_••.•.•••.•....•.••.._.•....••••...49

Introduction•.•.--.-.•...•.--.--...•.••.•.•.-...--.- ----.•....····---···50 Method••_••_._••••••••••_._••••••_••_._••••••_•••••_••_•••••_••••••_••50 Results _._._..••••••••••••••••-•.••••...•._._ _.._ __ -51 Discussion •••••_•••...••••••_•••.•••.-••••••.._--.-_..._._-_...----.56

GENERAL DISCUSSION••••...•••..•••.••••••••••.•••••••••.•••.•••51 REFERENCES •.•..•.•.•••..••••...•••..•.••••••••.•••••••••••••_..•···58

vii

LIST OF FIGURES

Figure 1: SchematicDrawingof The Hippocampus ••- -- - 7 FlQure2:DiagramofOCcludingDevice- -- - -- - _ 19 Agure3:Diagram ofOct luding Table -- - ---.-.- --_.__21 Figure4: OpenField ActivityFollowingRepeated lschemla---- 26 Figure5: PhotomicrographsofCA1 Areas ••••-.... _....--••••-•••--•• 29 Figure6:Degree ofCAlCell Loss •••--.-.-.----••••••••-•••-.-... .31 Figure7: OpenFieldActivity in Familiar and NovelTesting

Environments--- ••- - - -. - ••- ..-- -- - ••--__• 40 FlQure8: Photomicrographs ofCAlAreas- - -.--.--_ .- 43 Figure 9: Degree ofCA1 CellLoss - - --- - 45 figure 10:Open FieldActivityof GradedIschemia••- . - --- 52

Ftgurs11:Photomicrographs of CAlareas 57

FlQure12:Degree ofCAl Cellloss- -- .-- - - - -- - 59 FlQure13: ComparisonBetweenA.B.andIschemiaMOdal- - 64

viii

LISTOF TABLES

Table1:Comparison01locomotorActivity -- ._._ - _. -54

Ix

LIST OF ABBREVIATIONS

ANOVA _. Analysisof Variance

AMPA••..d-amino-:'3-hydroxy-S·melhyl-4-isoxazolepropionicacid AP4._._.z-arrmc-e-pncsorcncberyrate

APS •••••z-emlno-s-phcspnono-vatencacid AP7 •••••z-arranc-r-phcscnoncneptanctcacid CGS•••••cis-4·phosphonomethyl·2-piperidinecarboxylicacid CPP_._._. 3-(2-carboxyl-piperazin-4-yl)propyl-1-phosphonlcacid EM••••• ExcitatoryAminoacid

LTP••••• Long-term potentiation

MK·801-·(+)-5-melhyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,to-Imine maleate

NMDA•••N-melhyl-D-asparticacid MelD ••-.MicrocomputerImageDevices PCP•••••1o(1-phenylcyctohex yl)-plperldlne

GENERAL INTRODUCTION

1. Cerebral Ischemia

Clinicalfeatures01stroke

The goal of the presentexperimentswas to investigatethe relationship between cerebral ischemia.hippocampalpathology and functionaldeficitsin an animal model.Beforegetting10the animal model of cerebralIschemia, a brief reviewof clinicalstrokeis presented.

Stroke was recogni::iedas a group of neurologicalsyndromescalledIt

Zhongleng"in a traditional Chinese medicine book"Huang Oi Net Jing"(Canon of Medicine)about 500 B.C.This book describes the neurological syndromes caused by "the imbalanceof the blood In tho body as a result of catching acold wind",In Europe,SOranus of Esphesus(A.D.98-138)observedthe"hemiplegic paralysis"most!lften occurringin the elderlyduring winter(Porken,1974; Fields and lemak,1989).

Nowadays,strokeis one of the most commonlife-threatening

diseasesand is the third leadingcause of death in the UnitedStatesafter heart diseaseand cancer(Wolf et at,1986).The AmericanHeart Association (1963) reportedthat there are about500,000strokevictimseach year.Thetypesof ischemicstrokecan be identifiedby differentclinicalfeatures.Datafrom the populationof Rochester,Minnesota(Garrawayat aI., 1983),giveus an idea aboutmortalityratesamongdjff~rentstroketypes.In this report,the average annualincidencerates per 100,000populationfor specificstroketypes from

1975-1979are cerebral infarction (75).intracerebral haemorrhage (13), suba rachnoidhaemorrhag e (11), stroke ofuncertaintypes (4),andalltypes (103).10addition,manymedical disorderscancause strokes.Theseinclude, anoxicflSChemicdamage of thebrainfollowing:card iac arrest (Caronna and Flnklesteln.1978);cardiac surgery(Branthwaite,1~/2);traum a anddissection of cervco-cerebret arteries (Oragon etal.•1981).From the above itcanbeseen thatstrokehasahigh incidence,hll.Jhmortalityrate andmanycausal factors.In thesearchlortreatments01this complicated disease,manydifferent"stroke"

models have been examinedin t:,elast 150years.

ModelsofCerebral Ischemia:

1).COmpletelycutting theblood supplytothebrain-thismethodIs represented bydece plteucn.Brown-Sequardmadethe first observatio nsof this kindin 1858.He observedchangesotrespiratorymovement indecapitated dogs(Weinberger et al..194O).LaterHaym nnandBarrierrepo rtedthat automatic reflexeswerego ne after12 minutrs01cerebralisc hemia (Weinberg er atal.,1940).Allot tile functio nal observationsIn thedecapitated animalswerefocusedontheischemiceffect s onthe nervereflexes.respiratory, cardiacregulatoryandvasomotorcentres(Heymanset el.,1937).

2).Gener al cjrculatory~.this methodisrepresentedbystoppingthe generalcirculationpriortoreviving theanimals.Differe ntmet hodshavebeen used:chlo roformintoxication and resuscitationbyintraarterial lnjeclio nof epinephrine andcardiac massage(Crile and Dolly,1908);electricalshockto prod uce ventricularfibrillati on andcardiacmassage10restoreIhe normal heart beat;and haemorrhage orasphyxia (Weinbe rgeretal.,1940).

3).Occlu;;;ionof thearterialsl,!QQJy- Usuallythe carotidor vertebralarteriesor bothare occluded.Thismethodwas first introducedbyCoOP~I'in1836, in which heligatedboth carotidandvertebralarteriesinthe dog. Sincethen a greatdealof workhasbeen doneusing thisischemic model inclUding establishmentin otheranimals(GildeaandCObb,1930).

Inrecent years,thestudy of cerebralischemiahas tendedto use rodent models(GinsbergandBusto,1989).However, unilateralor bilateralocclus~onof the carotidarteriesdoes not produceischemicbraindamagein therat due to an efficientcollateralcirculation(Payanet aI.,1965;Eklof and Siesjo,1972).In order 10producecerebralischemiaInIhis an:mal,systemichypoxiaor hypotensionis required(SmithataI.,1984;Nordstrom and Rehncrona, 1977).

In 1979,Pulslnelll and Brierley Introduceda four-vesselocclusionmethodinthe rat byelectrocauterizing the vertebralarterythrough the alar foramina ofthefirst cervicalvertebraand reversible clampingof bothcarotidarteries.

4). Focalischemia- All oftheischemic modelsmentionedabove involveglobal ischemia.Recently,a methodfor occludlnqthe middlecerebralarteryhas been Introducedin whichthe arteryis occluded by a subtemporalcraniectomy (Tamuraet aI.,1981;Shlgenoet al.,1985;Bederson et aI.,1986).This model relatesmoredirectlytotheregional ischemiacommonlyobservedclinically.

The last two modelsmentionedabovehave some advantagesindifferent studies,but they arenotthe mostidealmodelin functionalstudiesdue to surgicalcomplications:suchas damageof the vertebralarteriesin the four- vesselocclusion modelandopen-skullsurgeryin the focal ischemiamodel.

Recently a gerbil model(describedbelow)of cerebralischemia hasbeen used quiteproductivelyin researchonstroke.

Mongolian gerbils,Mariones ungujculatus are rodents of the family Cricetidae . They are widely distributed in the regions of Mongoliaand northeastern parts of China (Rich,1968),In1966,Levine and Payan observed thatgerbil~~were susceptibleto cerebralischemia producedby occlusionof the carotid arteries (two-vessel occlusion).ThisIs because of the special cerebral-vascular circulation In the gerbil.Generally,thebrain is supplied by two pairs of arteries, the internal carotid and the vertebral.Th elatterfuse into thebasllar arteries at the base of the brain.The internal carotid artery dividesinto posterior,middle, and anteriorcerebral arteries.The two anterior cerebralarteriesjoin rostral to the optic chiasm.The basilar artery bifurcatesinto the two superiorcerebellar arteries. In humans and moslmammals.there are a pair of arteries.the posterior communicating arteries, whichconnectthe posterior cerebraland the basilar arterteato form a"comm unicating" bloodsupply at the base of the braincalled

"the circleof Willis"(Carpenterand Sutln.1983; Vamoriat aI.,1976). However.

there are no efficientconnecting arteries between the carotid andthe vertebrobasilar arteriesin thecircle of Willis Ingerbils (Levine and Sohn, 1969;

Kahn,1972; Harrisonetal.,1973).This makes it possible to produce forebrai.... ischemia in gerbilSby occluding thecarotid arteries.The gerbilmodel has features of simple surgery, fewer post·surgicalcomplicationsand effective ischemicdamagecompared with some 01the ischemic models mentioned before.

From the animalmodels of Ischemiaithas beenpossibleto classify the brain damage observedinto 2types:

1).Selective neuronal vulnt:lrability.The most extensive brain damage following ischemia is inthehippocampus,Specifically,neuronsin CA1areaare mostsensitive to ischemic insult(Ito at at, 1975;Kirino,1982).Sincethe

present experimentsfocusonthepost-ischemic functionaldeficits closely relatedto the ful'ion ofthe hippocampus,abrief review 01the anatomy and neurotransmittersofthe hippocampusispresented below,

2).Pan-necrosis.In this type the damagenot onlyeffects neuronsbut also the glialand vascularcells.Prolonging the duration of ischemia or irreversible occlusionof the blood supplywill transform selective neuronal damageinto pan-necrosis(Hassmanand Kleihues,1973; Kirinoandser e. 1964), To study more specificfunctional deficitswhichisonlyrelated to certainbrain structures this type of damageis avoided,

2.Neuroanatomy01 Hippocampus

The hippocampusinmammalsconsists primarily ofpyramidal neurons and associatedinterneurons.These neuronsare packedtogether inone layer of a three-layeredstructure,comparedto the six-layeredstructurein the cortex (O'Keefeand Nadel,1978).The hippocampus(Figure1)Is dividedintot~

majorparts,the fasciadentata (dentategyrus) and hippocampus proper (cornus ammonis).The hippocampusproper hasbeen further dividedintofour subfields ComusAmmo nis (CA)1-4(Lorente deNo.1934),CAl refers to the area"regio superior"whichislocated nearthe distalend of the dense plexus .CA2 and CA3 aremainly includedin theareacalled-regia inferior"which arelocated at the dentate end, eM stands for the pyramidalcells and interneuronsscattered insidethe hilusofthe fascia dentata.There aremanyhypothesesabout neurotransmissionbetween the differentregionswithin the hippocampus (Frotscher etal,1988).Inorderto get a clearviewaboutthis neurctranamission.a concisedescriptionofthe input,

transmissionand outputofthehippocampus (Zola-Morganatat,1966)isgiven here.Asshownin Figure1,thisneural circuitryfinishesasa closedloopinthe entorhinalcortex.Among the pathway smentioned above,there are three pathwaysusing excitatory amL"'IOack:ls(EM):1.The perforantpathw ayforms themainglutamalelaspartate - containingaff~entsin thehippocamp us (Fonnum, 1984);2.Themossyfibres terminatingIn thestratumIucid.Jm (White atal.,1977);3.The commi ssural fibres(contralateral)and Schafter colfaterals (ipsilateral) fromCA3whichterminate in CAl andCA3 (Cotma n and Nadler, 1981).

Besides the glutamatesystemthere are many hypothesesabout neurotransmissioninthe hippocampus,e.g.,cholinergicsystemand S·HT system etc.(Frotscheretal.,1968).Becauseithasbeen foundthatthe glutamatesystem has a dualrole inmemory andischemicneuronal death.the roleofthe excitatory amino acid isemphasisedhere.

3.Excitatoryamino acid s

Intheearly1960's,glutamatewasrustproposed asa neuroexcitatoryagent by Curtisand Watkins(1963).RecentlyEMhave been suggestedto be implicatedinlearning,memo ry andotherbrainfunctions.Inadditiontotheirrole as neurotransmitterstheEM are also neurotoxicat hIgh concentratio ns(Harris etal.,1984;Clineschmldtetal.,1982;Schwarczetal.,1984;Cotmanand Iversen,1987;Rothman and Olney, 1987;Monaghan etal.,1989).Within the last 10 yearsithasbecome clear thattherearemultipleEMreceptors,notjust the glutamatereceptor as originally thought.

FigureI.Schematic drawing of the hippocampus .

The linesshowtheunidirectional pathway(adaptedfrom Zola-Morgancrat. 1986).

(I).Entorhina lcortex(EC)(majorinput) I (pcrforant pathway) (2).Dentate gyrus (OG)

I (mossy fibers:excitatory) (3).CA3

I (Schatfercol latera ls) (4). CA l

I

(5).Subicularcortex(5) I

Entorhinalcortex.

Theexistenceof EM receptorsubtypeswas deducedby the relativeactions 01selectiveagonistsand antagonists(Cotmanand Iversen,1987).Based primarilyon agonistresponsethe EM receptorshave beenclassifiedintofive types(Watkinset

e..

1990):1) N-methyl-D-aspartate(NMOA);2)o-amlno·3- hydroxy·5-melhyl·4-isoxazolepropionicacid(AMPA); 3) kainate(K);4) 2-amino·

4-phosphonobutyrate(AP4);5) metabotroplc receptors. NMDA,a glutamateanalogue,isa selectiveagonistwhichhas receivedconsiderable attenton as severalfunctionshavebeen attributedto the receptor activated by this agonist (see below).Radioligand autoradiography showsthat the highestlevelof NMOAbindingsitesin the brain Isin the hippocampus (Monaghanet at,1963;Monaghanand Cotman, 1985;Mayer and Westbrook,1987).Withinthe hippocampus,the CA1 region showsthe highest levelof NMDAbinding Sites,whereasmoderatelevelsare found in CA3 and dentategyrus(Geddesetal.,1986;Monaghan and Cotman.1986).

AMPA,a structuralanalogueof quisquallcacid, Is morespecificIn binding studies thanqulsquatate(theoriginal agonistused to definethis receptor) (Honore et ai,1982;Monaghanat at,1989).The distributionof AMPAbinding sites in the centralnervoussystemis parallelto NMDAbindingsites.The functionof the AMPAreceptorinvolvesgenerationof fast EPSPsIn many central EM pathways(Monaghanetal.,1989;Watkinsat at. 1990).

Kainateis not as specifican agonist as NMDAand AMPA, and some responseto kainatemay be mediatedby AMPAreceptors(Monaghan et ai"

1989;Honore et aI,1982).However,the findingota pure populationof kainate receptorsoccurringon mammalianC-fibersIn the absenceof AMPAand NMDA receptorsprovidesthe evidence for discreteexistenceof kainatereceptors (Agrawal andEvans,1986;Watkinset at,1990).

10

Thelasttworeceptors,AP4and metabotrop icarenotas clearlyunderstood as th e former three recept ors,andworkisstillrequiredtochar acteriz e these receptors(Monaghan atat.1989;Walkinsetat.1990 ).

NMDAreceptors andlearningandme mory

One of themost excitingfindingsaboutEMisthe role oftheNMDAreceptor intheinduction of long-termpotentiation(LlP).lTPrefers10 aphenomenon in whicha short burst0' highfrequencystimulationresultsin polenliation ofthe evokedpopulation spike.This potentiationmaylast forhours or daysandis widely regarded asacellu larmod e l01le a rningandmem o ry(Bliss andLorna, 1973;Alg er andTeyler,19 76;CollingrldgeandBliss,198 7;Wroblewskiand Danysz.1989).

The role ofNMDAreceptors in theinduction ofLTPwar;firstdemonstratedIn th e CAl -eqio nofhippocampal slic e . LTPinthe Schaffer/co mm issuralpathway isreversiblyprevent edbyiontophoreticadministratio n of2-amino-5- phosphono-valericacid(A PS).a competitiveNMDAantagonist.into thesynaptic region(Collingridgeetal..1983;Harriset81.•1984:Collingridgeand Bli ss,1987) .Also. phencyclid ine(PCP)andketamlne .bothnon-competitlve antago nistsof the NMOAreceptorprevent the induction 01lTPin theCA l region(St ringerandGuyen at,1983).Morrisatal.(1986) foundthat chron ic Int rav entricular inject ionof AP5cou ldcau saa selective imp airment of plac e learningin rats.and APStreatme n t also su ppressedlTPinvivo. There area numberof otherstudiesimplicating NMDAinlearn ing andmemo ry.For example.otherNMOAantagonists.e.g.• 3-(2-ca rbo xy-,pipe razin·4·yl)propy 1-1· pho sphon ic acid(CPP).cis-4-phosphonome thyl-2. piper idine carboxylicacid

11

(CGS19755) and PCP,have been foundtoimpair perlormance on passive avoidance tests(Benvenga atat. 1989).(+)-5-methy'I-10,11-dihydro-5H- dibenzo(a .d) cycloheplen-S.10- imine maleate (MK-801),a non-compeuve NMDAreceptor antagonist,can produceimpairmentonthe radialarm maze and increaselocomotor activityinthe open field test (Shapiroand Caramanos, 1989;Heals and Harley,1990). It seems NMDA plays animportant rolein learningand memory.Unfortunatelythe same cetlularactionof NMDA receptors which appearimportant forlearningandmemory.canlead 10neuronal toxicity whenthe receptoris excessivelyactivated.

Ischemiainducedneuronaldeath

Manyhypothes eshavebeenput forthto explainthemechanism of neurona l deathfollo w ing ischemicepisodes .Neurotoxicity01excitatory amino acids providesoneof the most popular concepts aboutIschemiainducedneuronal death,The neurotox icityofEMwas first shown by Lucas and Newhousein 1957,who foundthatsystemicinjection of glutamate destroysthe inner neural layersof theimmature mouse retina.Sincethenmany studies onthe neurotoxicit yotthe EAA havebeencarried out (Olneyetat,1971; Olney,1978, Monaghan etal.,1989).Thesestud iesshow that systemicadministration of glutamatadestroysneurons of the brainin newbornmice,rats or monkeysin brain regions whichlacka blood brainbarrier.

The select iveloss of neurons is seenin regions characterized by extensive NMDA binding, and glutamate levels are increased in the hippocampusduring isc hemia (Jorgensen and Diemer,1982; Cotman and Iversen,1987).Some EMantago nists can protect againstischemic brain damage.Forexa mple, local

12

administration01 a selective NMOAantagonist 2-amino-7-phosphonoheptanolc acid (2-APH)into hippocampuseffectively preventsIschemicneuronal damage (Simonetat,1984);Systemicadministrationofother NMDAantagonists.e.g., MK·601,CG8-19755and riluzolehave also beenreportedto protect against Ischemicbraindamage,givingfurmersupportto the proposedtheory that excessiveNMOAactivationmaytrigger ischemicbraindamage (Gill91at.1987:

Margouris at at,1989).

4. Functional Studies

From theabove.it mightbe concludedthatstrokeis one of the most ilia- threateningdiseases.To study thisphenomenonmany animalmodelsof cerebralischemiahavebeenestablished.Inthesemodelsmostresearchhas focusedon the hippocampusbecauseit was themostvuherable toischemic damage.Becausethe hippocampusplays animportantroleinlearningan~

memory.it isimportantto understand the functionaldeficitsresultingfrom hippocampaldamage.

Scovillefirst reporteda caseof severeanterogradeamnesiafollowinga bilateralmedialtemporalloberesection(damageto the anterior2/3of hlppccampusand medialtemporalcortex)ina patient(H.M.)who sufferedfrom intractableseizures In1954.AlthoughH. M.wasextensivelystudied (Scoville, 1954; Scovilleand Milner,1957)and continuestobe stooled. hisdamageW'J.S clearly not confinedtothehippocampus.The conclusionthatdamagelimitej10 hippocampuscan causeamnesiawas not certainuntilthe casereport ofA.B.

by Zola-Morgan el al.in 1986.PatientA.B.sufferedseverelearningand memoryimpairmentsfollowinganischemic episoderesultingfrom coronary

f3

bypass surgery.Systematic memorytests performedduringthe liveyears precedinghis deathshowedthat A.B.exhibitedsevereanterograde amnesia.

almost no retrogradeamnesiaand no significantchangein his personalityand intelligence.Afterhe died,wholebrainsector-sweremade,and the most prominent lesionsinvolvedthe CA1 region of the hippocampus.Damageof the CA1region isthe only lesionthai could be found to explainR.80'samnesia.

In animalstudies,a varietyof teste havebeen useeto measurethe animals' spatial learning and memory (Barnes, 1988). Many recent studies have been performed to determinethe relationship betweenfunctional deficits(e.g"

learning and memory) and the hippocampusin ischemic models.An overview of the tests which have been used in testlnq learning and memory in ischemic models are described below :

1).MQrrjsWater Maze. Thistest was firstintroducedby Morrisetel.in 1982, the basic principle of Ihe lest is thai there is an escape plattcrm whichis hidden beneath the surface of water,made opaque wilh milk,;i1a fixed locationreialive tc visible environmental cues outsideth':l pool. Animals are released at random ccations,and after a few trials the normal animalslearn to swim directly to the hidden platform,wheraas animals with hippocampal lesions take a significantly longer time to find the platform. In this test animals are thouqht to use their reference memory ot the environment 10 locate the platform (Morrisat af.,1982).

Some studies haveshown thai ischemic damage limitedto half ofthe CAl sector of the hippocampus can be severe enough to Impairthe animals' performance onthistest (Auer etal.,1989).This finding gives furtherevidence for an imponanl role of the CAl sector in place learning and memory.

2). Radial Arm Maze- With thisexperimental paradigm, animals obtain IQod pellets in one of several arms, usually there are 81010anne.In order to get Ihe

14

food the animals have to use memory of the -environment- to locate the correct arm (Nagni at al. 1979). Two types of memory are distinguished in the radial arm test reference memoryandworking memory.Reference memoryrelieson the location of fixed externalstimuli (e.g.• location of a window) relative 10each lest arm.Thesecues do netchangefromtest day10testday.Workingmemoryrelers to trial bytrial changesin that the animal must remember which arms have been entered on a particular trial.It has been shown thai animalswith hippocampal lesions take longer to localethe rightarm than controlanimals(Olton 1978;

Barnes1988).Peelerand Smith(1990)showedthat 5-7 minutebilateralcarotid occlusionin gerbil whichproduced severe bilateralloss of CA1cellsled to significantlymore re-enlriesin the radial arm maze.

3).Passive AyoldanceTest- Eventhough this test is not a specificspatial memorytest,somestudieshave shownthatgerbilswithischemicdamageof hippocampus showpoor performance on thistest (Malgouris et at,1989;

Tominagaand Ohnlshl,S. T.,1989).

Differentfrom all the abovelearningand memorytests, Chandler et al.

(1985) introduced openfield tests in ischemic studies.Chandleret ere study showedthatthe mostprominentbehavioural changefollowing 5 minutesof forebrainischemiain the gerbil is a large increasein locomotor activity,evident atl4hrs postocclusion andgradually diminishing10normalafter about5 days. In a more systematic study,Gerhardt and Boast(1988)examined the relationshipbetweenlocomotor activityand degree of ischemic damage of the hippocampus In gerbils.In their studyischemicdamageof the hippocampus wasinduced by bilateralocclusion of both carotid arteriesfor20minutes.

Lo..:omotoractivitywas recorded onthe first and fourthdaysafterccckrslcn Histological assessmentrevealedneuronaldamage in the CAl areaof the

15

hippocampus.The meandistancethatthe ischemicanimalstravelled(as a Indexof locomotor activity)duringDay 1 andDay4 wasapproximately three- foldcompare jtothecontrolanimals.tIwas found that mere was a positive correlation between theposlischemichyperactivity andthe degreeof neuronal degeneration inthe hippocampus.

Comparingtheopenfieldtest totheother threetests mentioned above.it seemsthatopenfieldlest hasthe advantageof simplicity.However,it hasnot been established thatthe open fieldtestis aneffective funct'cnaltest in cerebral Ischemia experiments.Furthermorethere hasnot been a clearindication of the mechanism underlyingthe post-ischemicincrease inlocomotoractivity.Is the Increase In locomotoractivity a resultof a simple motor hyperactivityorsome other runetionaldeficit.lorexample,spatialle arnIng ormemorydeficit ?Inthe presentexperiments therelationship amongcerebralischemia, locomotor activityandspatialmemory wasstudied.

16

EXPERIMENT ONE

17 INTRODUCTION

Atpresent.most studiesof cerebralischemiain gerbilsutilize a one-episode Ischemia model.Afewstudieshave involved repetitive cerebralischemiain which the intervals between theepisodesof cerebralischemia werelimitedto a few hours (Tomida ataI., 1987,Vass etat, 1988. Katoatal.,1989). However, these studiesdid not examine any of the behavioural deficits resulting from repetitive cerebralischemia.Resultsofarecen t study (Gerha rdtand Beast.

1988) suggestthai the degreeofhip pocamp a l damage is posltlvely correlated withincreasedlocomo toractivity. Concerning therelat io ns hip between severity of the ischemia and the degreeof heightenedlocomotor activation,would more severe ischemicdamage, resultingfrom repeatedIschemicepisodesor longer carotid occlusiontimes,producecorrespondingIncreasesIn locomotoractivity?

In the firstexperimantamodel01repeatedischemia(Tomidaatat,1987;Vass etal.,1968)was used to address this question.

METHODS

SUbject s

Adult femalegerbils wereprovided byHigh OakRanchLtd, Goodwood, Ontario,Canada,andweighed 45-70g at thetime of surgery. The gerbltswere housed in plasticcages al atemperature of 22°C on a12·hrday/night cycleand fedwith commerclal pellets andwateradlibitum. All ofthe animalshadbeen residentIntheir cagesforabout fourweeksbeforetheexperimentsbegan.

18 Surgery

Animalswere anesthetizedby intraperitonealinjection of sodium pentobarbilal(50 mg/kg) followingpretreatmentwith an Intramuscularinjection of atropine(0.11flg/kg).They werefixed in a supine position and an Incision about1.5emin length was made inthe anteriorcervical midline. Both carotid arterieswerecarefully separatedfromthe surroundingtissue.A chronic occludingdevice was Implanted(Figure 2), suchthat the ends of the occluding and releasing threads were directedsubcutane ousl y10the backof the neck where they extended 1 emout of the skin.One week was allowed for the animalstvrecover from the surgery.

Prior to the occlusion,the animalswere anesthetizedIn a plastic chamber with ether,andthe anesthesiawas maintainedby an-elher mask-(the open endof a syringe whichwas filledwithether-soakedcotton).Occlusionwas accomplished by bilateraltighteningof the occludingthreads and was terminatedby bilateraltighter;~.•l1of the releasingthreads (Figures2 and 3).

Animalswerekept under a 60 w lamp (SO em in height) fromthe beginningof the surgery until the animalrecoveredfrom the anesthesia.

Procedure

Twenty-threeadult female gerbilswere usedin this experiment.In the ischemicgroup(n ::12),daily 10 minuteopen-fieldtests were begun?4 hrs afterthe occlusionfor 4 successivedays In the first week.In the secondweek the same proceduresof occlusionand open field tests were performedagain.

19

Figure2. Diagramof OccludingDevice

The occludingdevice asdescribedbyToroida et al. 1987.The implantation procedurewasmodifiedbythreadingtheoccludingand releas ingthreadsthroug h the devicewiththeaid of aloop of #6.0silk suture.

20

~ ^{. . .} L"" """

~~.:' ~"'''~

Lull (21

21

Figure 3.Diagram of OccludingTable Theocclusionwasmadebybilateraltightening of theoccluding threads (Occ)maintainedbytwo weights( about80grams) hungateach each end of theoccluding threads.The occlusionwasterminatedby bilatcrallight ening ofthereleasing threads(ReI).

22

ReI.

23

After finishingthe second week ofopenfield testing,fouranimalsfrom this group wereperfused forhistology.A thirdocclusionwas performedon the sevenanimals remaining inthe group.These animalswere perfusedfour days afteropenfieldtesting.

Inthe controlgroup(n=5). thesameprocedures ofsurgery wereperformed but without occlusion.The open fieldtests were carriedon for threeweeks (four test days perweek).

A singleocclusion(10minutes)was performedinsix animals.The animals were perfusedfive days afterthe occlusionwithoutbehaviouraltesting.This group was used 10examinethe histologicalchangesafter a single ischemic episode.

Open-tl eld test s

Open-fieldtestswereperformed in a woodenbox measuring75 emx 75em x 50 em.The floorwas dividedInto25 equalsquaresof approximatly 15 em x15 em.The box wasilluminated by two 60 Wlamps positionedabout 1.5meters abovethe floor.A viduo cameramounted over the box was used for recording the activityof theanimals.Thenumber of squaresthe animalsenteredin 10 minutes was countedas themeasureof locomotoractivity.

Histology

After the openfield testing was finished,the animalsweretranscardiajy perfused with 20mlof 0.9%heparinized salinefollowedby 30mlof 10%

bufferedformalin.Thebrains were fixed in10%formalin atroomtemperature

24

ovemight.they werethencutinto 40 urncoronalsections at·230C ona freezingmicrotome.Sectionswere takenfromthe most rostral lip of the hippocampus to5.0mmposterior 10bregma.sectionsweredehydrat edwith grad ed ethanols and stained withcresyl violet.

A MicrocomputerImageDevice (Me lD:ImagingResearchInc.Brock University,St.Catharines.Ontario)was usedtoassess damage ofthe CAt regionofthe hippocampus.The basicprincipleof MelDis tomeasure the area of a brainstructureusing the relativeopticaldensity.Therelativeopticaldensity ofIhedentate gyruswas chosen as the targetvalue (similar10normalCAt relative opticaldensity),andthe relative optical density of the molecular layer ventraltoCA1waschosenas the backgroundvalue.Thethreshold valuewas determined as follows:

ThreshOldvalue-(BV•TV)1O.4+ TV BV-Backgroundvalue TV-Target value

The threshold valuewas criticalin measuring theCA1area,becauseonlythe relative opticalden~ilYlevelabove the thresholdvalue couldbedetected.In this experim ent. thesectionsforMCIDanalysis corresponded to a plane 1.6mm posteriortobr8£ma.The CA 1region fromits medial tolateral boundary was delineated and thearea exceedingthe threshold value wasmeasured.The mean area(mm 2) of both rightandleft hemisphereswascombined to yield a finalscore.

25 RESULTS

General obs ervations

Abnormalbehaviourwas not evidentpost-lschemicajly, and the animals' food and waterintake appearednormal (no weightloss in post-lschemlc animals),No adversesymptomswere observed In animalswiththechronic occlusiondeviceduringthe 3 weektesting period.An autopsyperformedon oneanimalat the endofthe third testing week shOwedregenerative tissue surroundingthe device andnoinfection was observed.Thedevice was situated in the originalpositionand workedperfectly.

Locomotor activity

As showninFigure 4.locomotoractivityofthe ischemiagroupincreased after thefirst episodeofIschemia IF (1,15)=12.0,P<O.Ol,ANOVAj.and was significantlyelevatedrelative to the controlgroupforthefirs!three days (p<

0.01,Dunnett's Test).The second andthe thirdocclusions lailed toIncrease locomotoractivity above controlvalues(P>0.05,Dunnett'sTest).Locomotor activitywasmaximal 24 hours afterthefirstIschemia, and was significant compared 10the 2nd,3rdand thelollow ing test days (P<O.Ol, Dunnett's Test).

The levelof locomotor activityof the control groupremainedrelatively constant over testingdays while thelocom otor activity ofthe ischemicgroupdropped sharply,especiallyafter the first twotest days. Thisbehaviouralpattern yieldeda significant Day effect (F (3,45)""28.4,P<.00 11and a TreatmentX Days interaction[F (3,45)=11.6,P<0.01].

26

Figure 4. OpenField ActivityFollowing Repeated Ischemia

Openfieldactivityexpressedas squaresenteredper 10mintest session for the Control(C)andthe Ischemia (I) animals.DaysJ..4- represent themean activityscores.:tSEMfor theIx1animals.Days5-8 forthe Ix2animalsand Days 9·12for theIx3animals.

27

700

1stoeet,

600

500

<>

'0

e

400

S

c W

.. _e

•

:> 300

<T 1Il

200

100

0

0 2 3 4 5 6 7 8 9 101112

DAYS

28

Histology

Previousstudieshaveshown the CAlsectortobe mainlyresponsibletor post-ischemichyperactivityand spatialmemory defICits(GerhardtandBoast, 1988;Auer atat,1989).Therefore.in thisexperimentandthe othertwo experiments.onlythe CAlsector was examined.It appearsthaICAlregion was progressively damaged alter eachIschemic episode(Figure 5).

Figure6 illustratesthe computer-generatedmeasurementofCAlcell loss In eachofthe experimentalgroups compared tothe controlanimals (F(3,18)""

5.B6,P<0.01).FromMelDresults,some comparisonsof C/\1area(mm2) were performed.

eM

areasin the two time (I)(2)and three time(I)(3)Ischemic groupswere severelydamagedcomparedto the controlgroup(ConlrolVs.Ix2:

p<0.05;ControlVs.1x3:p <0.01;Dunnett'sTest).Ho'ft'~er,CAtsectorwas less damaged in the 1x I group,and did notreach statisticalsignir~f'Ir;A(P>

0.05,Dunnett'stest). There was no statisticallysignificantdifference in damage among I x3 Vs.Ix2andIx2v«Ix1 (P>0.05). ExceptIx3v«I xt was statisticaJtysignificant(FISherPLSD,P<0.05).

DISCUSSION

In thisexperiment,locomotoractivitywas transientlyIncreased after thefirst ischemicinsult,which Is consistent withearlierobservations(Chandleret aI., 1985;Gerhardtand Boast,1988).However, additionalischemicdamage resultingfrom the secondand third occlusions did notfurtherincreasethe activitylevel,which is notconsistentwith the conclusionthai there is a positive relationship betweenlocomotoractivityandCA1 damage (GerhardtandBoast,

29

Figure S.Photomicrographs of CAlareas

Representivephotomicrographstakenfrom Control(A),1x1(8 ),1x2 (e) and Ix 3 (D) animals. Note theprogressiveloss of pyramidalcells with eachsubsequentocclusion(B-D).

30

.. ' " ·f

.. 0' ,_-,

,

1

DOAIm

31

Figure6.Degr eeorCAICellLoss CAlarea(mm2)in the controland ischemicgroups.Eachbar represents the mean area±5EMpooledfrombothhemispheres.

< o

32

0.3

0.2

0.1

0.0-W-_-.1....L_ _.t...L_-.L...L_-..L.

Control I x 1 I X 2 IX3

33

1988).However,one main differencebetweenthe present experimentand Gerhardtand Boast'sstudy is that the hippocampaldamage In the present experimentwas producedby repealedischemia episodes, and in theirstudy was producedafter a singleocclusion.

Severalquestionswere raised by this experiment:

1).Why did the first 10 minute occlusionnot resultin significantCAl damage,and why werethere some variationsin the degree ofdamage of CAl even in the same ischemicgroUfJ?

2).Why didlocomotoraClivityincrease only after the firstischem icepisode and not after repeatedischemic episodeswhen hippocampaldamagewas more extensive?

3). What Is the mechanismunderlying these changes?

Topartly answerquestion 1. one has to go backto the methodsemployed.

Thisexperimentwas designed before it was appreciatedthat hypothermiahad a protectiveeffect againstcerebral ischemia.Thus, in this experimentliltle attentionwaspaid to bodytemperatureand the"cooling effect"from the ether anesthesia.This could account for the relativelyminor loss of CA1 after a single occlusionand the variation in the degree of damage of hippocampus,because other experimentshave shown that loweringthe body temperatureone or two degreescan allenuate ischemicdamap,e (Bustoat al., 1987;Corbettel at. 1990).

Forquestion 2. hypothermiamight also explain the significantdropin locomotoractivityover the first three post ischemicdays and the failure of the secondand third occlusionto Increaseit. The first period of Ischemiacaused incompletedamage of CA1 due to hypothermia.Thus, thq many functioning CA1 neurons[ef! after the firstischemic insult may have allowedthe animalsto

34

form a spatialmap of the openfieldenvironment.ThisIsfurther supportedby thefact thaItheanim alsgradu allydecreased theirlocomo tor activityover the first fewpostischemictest daysanddroppedtothecontrollevel after fourdays.

SubsequentIschemic episodeswhichcausedmoresevere ischemic damageof thehippocampus wouldnotbe expectedtoaherthelocomotor activitybecause theanimar smemoryor maphad already beenform ed(seeGeneral Disc ussion).Themap ones formedis thoughtnot10reside withinthe hippocampus(Squire,1986;2ola-Morganatat,1986.).

The aboveresults suggestthat theincrease In locomotor activityafter cerebralischemia(Chandleretal.,1985;Gerhardtand Boast,1988;Present results) maybedue tosomedisruption of spatialmapp ingability.In order to fu rt her lestthishypothesis.a secondexpe rimentwas design edin which the animalswerepre-exposed to the testenvironment prior to theischemia.

35

EXPERIMENT TWO

36 INTRODUCTION

Asnotedin thefirstexperiment,repealedischemicepisodesdidnot further Increaselocomotor activity,perhaps becausethe animal hadformeda spatial mapofthetest enviro nment Ifso,thenpre-exp osure to theopen fieldpriorto Ischem ic Insultshould blockor anenueteincre ased locomotor activitysince a 'spattal map"shouldhavealreadybeenformed.

In the prev iousstudyitwas foundthat10 minuteocclusionwiththeoccluding device wasinsufficientin producingeffectiveCA1damage,soin this experim ent the procedu re wasmodifiedin two ways:1) In order to avo id protec tive effects of hypothermia allsurgicalprocedures werecarriedout at 37.5⁰C;2) Sinceonly asing leocclusionwas to be performed, small mlcrovescular clampswereusedinst ead of the polyethyleneoccludingdevice thathad been usedin Experiment1.

METHODS

Subjects

Fort yadult female gerbils wererandomlydivided into three groups: ischemia (GroupI.n=10).pre-exposureischemia (GroupFIN.n=10).pre-exposure surgery contro l(GroupFSN,n=10).andsurgery control (GroupS,n=10).

Surgery and occlusion

Anesthesiawasinduced In a small plasticchamberwitha mixtureof oxygen:

37

nitrogen:halothaneat a flowrate30%:70%:2.5%(mllrnin.).Theanesthetic was deliveredthrougha F1uot8CAnestheticMachine(ForeggerCo.,Inc.Roslyn Height,NewYorX.U.S.A).Theanimalswere thenfixedIn a supineposition andhalothaneflowratewas reduced10 1.5%.

Atwoemanteriormidline cervicalincision wasmade.andbothcarotid arteries werecarefully separatedfrom the surroundingtissueand the sympatheticnerves.Fiveminuteocclusion01thecarotidarteries wasmade by clamping thevessels withSchwartz mlcro-serreffnea (FineScienceToolsInc., NorthVancouver, B.C).Aftertheocclusionthemlcro-serreflneswereremoved simultaneouslyfromboth carotidarteries.Aftermakingsureof good reflow in botharteries,the animals' Incision was suturedandtheanimalswereplaced undera 60wlamp IntheIrcages.Thebodytemperatureof the animalswas maintainedat37.5⁰Cby a temperature.controlledblanket(HomeothemllC COntrol Unit482,Harvard ApparatusUd,Edenbride, Kent,U.K)throughout surger)'.

Open-fi eldtest

Apparatus

Open-fieldtesllngwasperformedin the same lesting apparatus asin Experiment1.An imagetrackingsystem (VP112 scanning unit.HVS IMAGE, Hampton,England)coupled toaTatung·7000 computer was used to countthe numberofsquaresthatthe animals enteredduringthe testingperiod.

38 Testing

In GroupI andGroupS,daily10minuteopen-fieldtestewere carried out 24 hrs afterocclusion/surgeryand continuedfor 10successivedays.

IntheGroupFINand GroupFSN.the daily10minuteopen-fieldtestswere startedfiv edays belorethesurgery.Merfive days oftesting,theanimalsInthe FINgroupweresubjecte d10a5minute bilateral occlusionofthe carotid arteries.For the FSNgroup, the carol1darte,;"swereIsolatedbutnot occluded.

Open fieldtestswerebegun24hrs afterocclusion/surgeryandco ntinuedfor five days.After this five daytest period,theactivityofthepre-expo sedgroupsIn a seml-no v elenvironment wasexam ined.The test environ mentwasaltered by inserting an ellipticalwhite plasticlinerintotheopenfieldtherebyobscuring the white andnaturalwoodwallsoftheopenfieldlest bOx. The floor wascovered witha piece ofstyrofoam.Only onelightwas used and its positionrelative to the testapparatus waschanged.Picturesandotherobjectswerehungonthe wallsctthelestingroom,the csilingwascoveredbycoloured picturesand the videocamerawas wrappedInawhitecoat.In addition,noiseprovidedbya large fanwaspresenton noveltestdays.Animalsof theFINandFSNgroups were then testedinthis-nevertest environmentlorfiveadditionaldays.

HIstology

Animalsinthefour groupswere sacrificedabout threeweeksafte rocclusion.

The proceduresof perfusionandhistologicalpreparation andMelDanalysis werethesameasinExperiment1.

39 RESULTS

Locomotor activity

Thefour experimentalgroups(Figure 7) consistedof two ischemiagroups(I and FIN groups) and two controlgroups(8 and FSN groups).

As expected,5minutes ofischemiaproduced alarge increaseinlocomotor activity as illustrated by the performance ofGroup Ianimals.locomotoractivity of theIgroupwashighlyincreased after the episodeofischemiarelative tothe Sgroup IAtwo-factorANOVAwithrepeated measures: F (1.16)::75.1. P<

0.001). Loco motor activityoftheI group animalsdropped to alower levelby the fifth day,butthelevel of locomotor activitywas significantlyhigher than thai ofthe 5 groupeventen days afterthe occlusion (FromDay5toDay 10,IVs. S, P<0.0 1, Dunn ett's Test}. Thelevel of locomotoractivity ofthe 5group remaine d relatively constantcomparedtothegradual reductioninlocomoto r activityof theI group(ove rthe firstfive testingdevs,there wasasignificant GroupXDayinteraction:F(9,144) =11.9,P<0.001).

The locomotor activityof theIgroupgraduallydecreasedover the first five testingdays, whichresulted ina significa nt Day effect (F(9.144)

=

16.0 , P<

0.001] . Pre-e xposure to theopen-field prior to ischemia(Group FIN) virtually blocked the post-ischemicincreasein locomotor activity.Although theIandFIN groupsunderwentthesameischemicins ult, thelocomolor activity ofthe Igroup was significantly higherthan that of the FINgroupin thefamiliarenvironme nt [F(1,16 ).. 39 .6,P<0.00 1). Againthere was a significantinteraction(F(4,64 )""

6.2,P<0,001] and significant Day effect [F(4,64)"" 15.2,P<0,001).Locomotor activity of the Igroup was highlyIncreasedcompar edtothat of the FINgroupon

40

Figur e7.Open Field Activityin Familiar and Novel Testing Environment

Daily meanj,Locomotorscoresof: 1). The ischemic animals( Group I).

2). The surgerycontrolanimals(GroupS),

3).The pre-exposedischemicanimals( Group FIN).

(Familiar Environment+Ischemia+Novel Environment) 4).The pre-exposedsurgerycontrolanimals(Group FSN).

(Familiar Environm ent+Sugery+No velEnvironm ent)

41

1200

..,

Cll

; 600

W

c

III

2! co

6- 400

l/)

o

...

1000 N ^T

800 • 1 r

- 8

-

200

2 3 4 5 6 7 8 9 10

DAYS

42

each of thefive test days in the postlschemjc familiarenvironment(from Dayt 10 Day 5.P < 0.01,Dunnett's Test).

Therewas nosignificantdifferencebetweenthe FIN and FSNgroupsover the first fivetestingdays(F(1.~6)=0.3.P>O.5)even though the FIN gerbils had beensubjectedto bUateralcarotidarteryocclusion.Thelocomotor activityof the FIN group increased slightly over the firstfew days alter ischemia relativeto the FSNgroupyielding a significant interaction (F(9,144)=3.7,P<O.OOI]. The locomotor activityin the novel environment was initially increased which produceda significantDayeffect(F(9,144)

=

16.5.P <0.001).

in theFIN group,comparison of the locomotoractivityin the three testing periods(5 dayspre-ischemia,5 days post-ischemiain familiarenvironment. 5 days post-ischemiain novelenvironment) showed thatthere wasno significant difference amongthesethree periods[F(2.27)=0.5.n.s].

HistologV

Severe neuronalnecrosisandcell loss (Figure B)could be seen in the CAl areaof hippocampusin bothI and FIN groups.TheCAl sectorwas severelydamaged in bothischemic groups[I and FIN Vs.S andFSN;F (2, 27)

=20.8,P<.001J.Individualcomparisons between groups showedsignificantcell loss in the I andFIN groupsrelativeto their respectivecontrols(P < 0.01, ScheffeF~Test).Therewasno difference in damage10CAl betweenthe FIN and I groups(Figure9),

43

Figure8.Photomicrographsof CAl Areas

RepresentivephotomicrographsoftheCA1 areafromasurgerycontrol (groupS)animal(top)andfromanischemic (groupI)animal(bottom).

Magnification160 X.Cresylvioletstain.

44

100,um

45

Figure9.Degree of CA1Cell Loss

Menuareaof CAl (mm2).±.S. E. M.inthecontrol(C ) and the ischemic groups (groupIandgroupFIN).

'" P<0.01.ScheffcTestcompared10control.

,...

o

c(

c(

WII:

c(

0.3

0.2

0.1

46

•

• T

T

0.0 c F + I + N

47 DISCUSSION

In the present exp eriment,theIand FINgro upsunderwe ntthesame ischemicinsult,however, the I groupshoweda significant increase inlocomotor activ itywhitethe FIN group showedactivity lev els comparable tothe cooperated control animals.Pre-exposure experience may thushave allowed the animalsto forma spatial map ofthe environment. Thus.for theFINgroup ischemicdamageofthehippocampuswouldnothavelao much effect onthe locom otor activitybecause ofthe pre-acquire dspatialmap.Thehipp ocampus is though t10 be a stationforinformation-processing(Squire, 1986 ; Zola-Mo rganet at.1986).The processedinformation(l.e.•a spatialmap 01theopenfield) would be storedin otherbrain structu res,suchas the cortex (Squire .1976;

Zol a-M organ etal.,1986).

Onewouldhave expected theFINgroupto show incr eased locomotor activity inthe"novel" environment. However,therewasno significa nt differ ence among theactivity scores recorded duringpre-ische mia,post-isc hem iaand

"nov el"enviro nment,whichmaybedue tothe'neverenvironment re allybeing

familiarto the anima l.Althou gh ,anu mber ofstimuliinthetesting room were changed,a great manyextern alcues (e.g.,odo ur,lab shelves,videocamera etc.)remaine d constant the rebydecreasingtheMnovelty" of the novel envi ronment.

Onefindinginthis experi menllhatdiffered from previous studies(Gerhard t andBoast, 1988) and from Exp erimen t1was that the level of locomo tor activ ity of theIgrou pnever dropped to the control level,remaining significantlyhigher than thatofthe Sgroup over all ten post-ischem ic test days.Thismaybe exp lainedby two facto rsin this experi ment whichare diffe rentfromExperiment

48

1:first,body temper atureoftheanimalswasmaintained Ihrough-o utthe occludingprocedures. so thaitherewasno-hypot hermi a- protection;the secondfactormaybethatthe microvascularclipsprovid eda moreeffective occlusionofthecarotidarteriesthant:idthe occludingdevice usedin Experiment1.Thus,Ischemicdamageof CAl wasmuchmoreseverein this experim ent than that in Experiment1. Inordertodeterminewhether this increase in locomo tor ac tivitywas proport ionalto thedegree ofisch emic damage, Experiment3wasdesigned.In this experiment.a differingdegreeof damageto CAl wasproducedby varyingtheduration of occlusion.

49

EXPERIMENT THREE

50 INTRODUCTION

As mentionedinthe general introduction, in Gerhardtand Boast's study (1988),the analysisof the different degreeof ischemicdamageof the hippocampuswascarried out Inthe same ischemic group(all Ischemic animals underwent a20 minuteocclusion) .Also in Experiment1, thecomparison of hippocampaldamage wascarried outusing a modelof'repeatedischemic episodes".In thepresentexperiment, the relationbetweenthe degreeof CA1 damageproducedby varying the occlusion duration and locomotor activitywas examined.

The purposeof thisexperimentthenwas 10see whethermore severe ischemicdamagetoCA1 wouldresult in gradedincreasesin locomotoractivity.

METHODS

aubtects

Thirteengerbilswere randomlydividedintotwo groups:10 minuteocclusion group (n :::: 8)and 15minute occlusiongroup(n::::5). Thedata fromSand I groupsof Experiment2 wereaddedto the data collectedfromthisexperiment.

Surgery and occlusion

The surgicalandanesthetic procedureswerethe sameasin theprevious experiment,exceptfor the occlusiontimes (10 minutesand 15 minutes),

51 Ope n-fie ld lest

Daily 10minute open-fieldtests were performedforlendays foreach group.

Themethod ofobservationoflocomotor activitywasthesame as in the previous experiment.

His to log y

The preparationwasthe sam e as in the previousexperiment.

RESULTS

Locomotor activity

As showninFigure10 andTabl e 1,Locomotoractivityof the ischemia groupsincreasedafterocclusion [F (3.29)

=

13.8.P< 0.001].Locomotoractivity of the5minute occlusion group (15) was significantlyincreasedcompared to the conlrolgroupIDunnet'sTest:Day l-Day6,P<0.0 1;Day7· Day10.P<0.05].

The locomotoractivityofthe10min ute occlusiongroup(110)and15 minute occlusio n group (I 15) wassignificantly incre asedcomparedto thecontr ol gro up fromDay1toOay 7 (P<0.01) andDay6 (P<0.05) andIromDay1toDay8 (P<O.Ol) respectively.AmongIhe ischemiagroups locomotoractivityof115 group wasincreasedsignificantlyon 3(Days 1,4and 5)01the nrst5 lest days [115VS. 15 and110.P<0.01).Thelocomotoractivity01the ischemic groups dropped sharply,especiallyforthe115 group(a significantTreatment XDays interaction:F(3.9)""43.0,P<0.001). All four groups showeddailydecreases

52

Figure 10.Open Field Act ivityof GradedIschemia

Openfieldactivityexpressedassquaresenteredper 10minutetest session. The dataofcontroland 5minischemia groupsis from experiment2.

54

TableI.Compa risonof locomotoractivity

Controlvsischemic groups(IS,110.115),comparedby Dunnett'sTest.

15 110 115

Dayl--Day6

Dunnett'sTest:

Day?

"

Day8 Day9

n.s Day10

.... - P<O.OI

• - --P<O.Os n.s- nC'?significant

56

in locomotoractivity (asignificant Day effect: F (3,27)

=

5.2,p"0.001). Hist ology

Asshown in Figure11 and 12, CAl necrosiscanbeseen inallischemic groups.Aone wayANOVAshowedsignificantCAl call loss IF (3.29)"" 28.7, P<0.001).Subsequent comparisonby Dunnett'sTest revealedsignificant CAl cell [assIn each ischemicgroupcompared10control(p <0.01).However,there was noclear differenceinseverity of damage to CAl amongthe threeischemic groups(P>0.05)except15 Vs.115 (Fisher PLSD.P<0.05 ).

DISCUSSION

The post-ischemic increase inlocomotor activityafter 10 and 15 minute occlusionswas similarto that observed previouslywitha five minute occlusion.

The locomotoractivity of the115groupwas increased significantlycompared to the15 and110 groups on Day 1, Day 4 andDay 5 but notonothertest days, whichmaybedue to the small numberof theanimalsIn the 110(n;::8) and 115 (n :::5) groups.This may reflectthe somewhatgreaterCA1damage sustained by the 115group.However,itwould seam thatchangesinlocomotor activityare near theirmaximallevelaftera five minute periodof Ischemia.The reason for this"limited increase"of locomotoractivity may be due to "functional specialization"within CA1, e.g.,rostral half of CA1 sector.Thus more extensive damage of CA1 sectorwill not have much furthereffect onlocomotor activity.

Another possibility Is thatthe leveloflocomotor activity Isnear themaximallevel for normalexploratorybehaviour,In otherwords,a behaviouralceiling effect.

57

Figure l l. PhotomicrographsorCAI Areas Representive photomicrographsof theCAI area fromalOminocclusion animal(top)and a15minocclusionanimal(botoro).

Magnification160X.Cresyl violetstain.

58

10 0

urn

Figure12.DegreeofCa ]CellLoss

MeanareaofCAl(mm2);tS.E.M.inthe control(C)and [he ischemic groups(15.110and115).Thedat a of controland15is fromexperimentZ.

60

0.3

0.2

o

^{Are aCA1}

<

U

~~

.«

0.1

0.0-l-l-_--JL,...J._ _.L.-JL..._.L.-L-_--'-

Control 15 110 I 15

61

GENERAL DISCUSSION

Extensivedamageofthe CAlregion of the hippocampuswas successful ly achieved in the pres entstudies.Theseresults furthe rconfirm thetwo-vessel occlusionin thegerbilas an effective,convenient ischemiamodel for stroke research(LevineandSohn.•1969: Kahn,1972; Harrisonatal.,1973 : Ginsberg

~ndBusto,19B9 ). In Experiment1,animalssubjected torepeatedischemic attacks didnotshowa graded increaseIn their locomotoractivity.Thiswas explained by the f9,clthalrepeatedischem icepisodeswould notbe expected to changea preacquiredspatial mapformed afterthefirstischemicattack (not severeenough duetotheprotectionby hypothermia) . In Experiment 2,pre- exposure10the ope nfield prevented thepost-ischemichyperactivity activity.

This was akey observationsinceitsuggestedthat theincreased locomotor activityinthe openfieldtestwas aspatialmemory deficit rather than a motor hyp eractivity.In Experiment 3,itwas foundthat more severeischemic damage of CA 1produce d bylonger occlusiondurations(10ancI')minutes)did not increaselocomotor activity substantiallyabove levels producedby standard5 minuteocclusion.

Fromallthree experimentsit was foundthat therewas a sensitive relatio nship betweenthe hippocampu s. sp allal memory, ischemiaand locomotor activity.Itshould be interesting to seeho w these relateandaffect eachother.First, one questionthat shouldbeclarifiediswhet herspatial memory deficitsrepresenta sp ecialfunctio nal deficit oraproblem withgeneral memory?In orderto answer this question,the brainstructure sinvolvedin spatialmemorymustbe known.Since differ entfeatur es of spatialanalysis are

j .

62

pertormedbydifferent cortical systems,theinformation mustbebrought together.Thestructurewhichencodes the relat ionshipbetween the information andthecortical systemisthought tobethe hppccampus (KOIb and Whishaw, 1990 ).Thus,patients withonlyhippocampaldamage do notpassin formation on to the cortical system,andshould onlyshowsymptomsof topographical disorientationrathe.than general memoryimp ai rment (Zola-Morganetat.

1986;Kolb and Whishaw,1990).Thismay weltberepresentedbythe case of H.M.HisIQis aboveaverage(118on the WechslerAdult Intelligence SCale).

Heisquiet and wellmannered socially. However, thefollowing description is evidence01spattaldeficits in H.M.••- After leavingthemain highway,weasked him for help inlocatinghis house. Hepromptly and courteouslyindicated to us several turns,unlitwe arrivedat a streetwhichhe said wasquite familiarto him.

Atthe same lime,he admined that we werenot at the right address.Aprone call to hismotherrevealedthatwe were onthe streetwhere he used tollve beforehis operation-- (Milner et at,1968 ).

It usedtobethought that H.M's spatialproblem resunedfrom his severe memory problems because heh~·jcombinedlesionsofthe amygdalaand hippocampus.However,as mentionedabove.H.M.has good memoryof the environment which hewasfamiliarwithbefore hissurgery, and onlyshows severe anterog radeamnesia.Therefore,H,M.'santerogradeamnesiais not theresu lt of a genera l memo ryprob lem.Recently,studies onA. B.showedthat ischemic damage to asmallportionofhippocampu s, the CA l reg ion ,brought onsevere anterograd e amnesi a (Zola-Morgan etal.,1986;Kolband Whish aw, 1990).

In thepresen t experjmeo ts,animals did notincreaselocomotor activity in the environm entwhichwasfamiliar beforeth eischemicepiso~e,but onlyshowed

63

increased locomotoractivity in a completely novel test environment (e.g.,group I in Experiment 2),which may be also called an"a nteroq rad e topographical amnesia".Comparingthis with the case of R.B(Figure 13), lt can be seen that amnesiabothin R.B.and the ischemiamodelhas a similarmechanism :CA1 loss,and the CAl loss can cause anterograde amnesiaor anterograde topographical amnesia.

So faritis clear that ischemic damage 01CA1 can cause anterograde topographical amnesia.In the present experiments,iflocomotor activityis taken as an Indexof spatialmemory. itis necessaryto knowhow spatialmemoryand locomotoractivityare related.In addition, itis also necessary to discussthe choice of the open-lieldtestas opposed to a radial arm mazeor Morris water maze, which are Ihought 10be "purer" tests of spettal memory.In order 10gel a clear understanding,Ihe concept of"environmental psychology"(WOhwill, 1970)has 10be introduced here.The ongoing activityof any animal is a complex mixtureofdifferentactivities,for example, walking,drinking ,eating, sniffing,sleeping,grooming, etc.Each species of animalengagesin a certain mixture of activitieswhich make them differfrom other species,and the reactions to particular external stimulationmay vary from species to species.

However,there arecertain patterns of behaviour that remainconstant across species. For example, in the situationof threat or stress most animals react by fleeing to safety (O'Keefe and Nadel,1978).The reaction of the animals depends on the novelty of the external stimuli. Noveltyelicits a state ofanxiety, and explorationis motivated by this slate.Spatial informationobtained during exploration will reduce fear and habituation to Ihe external stimulationwill develop (Halliday,1968;Groves and Thompson,1970).In the open-field test,

64

Figu re 13.Compa r ison of R.B and IschemiaModel Itcan be seenthatbothanterogradeamnesiain R.B.and anterograde topographical amnesiainischemiamodelare causedbythesame mechanism :CA 1loss.

65

66

the environment is novelto theanimal.The animalexplores the environment and exhibits increased locomotor activity until a spatialmap is formed (habituation). Threat,stress,food. wateranda sexualmateare thoughtto bein the sameclassof externalstimuli(O'KeefeandNadel.1978).Animalstestedin an open-tleld lest (stress of novel environment),Morriswater maze (stress 01 water)or radial arm maze (food deprivation) would undergo the same class of externalstimulation. Therefore. a similarspauetmapping processshould be utilized in all three tests. The open-field test shouldbe considered as testing an aspect01spatial memory, and post-ischemic changes of locomotor activity could beused as a sensitiveindex to examine the animal's spatialmemory.

The advantagesof the open-fieldtest areits simplicity,easeof observation and analysiscompared to the water maze.Moreover,the animalsdo not haveto be tooddeprivedas in the radialarm mazetest. It alsoallowsearl) testing after the

isch emic insult. Previousopen-tteldstudieshave notgivena clear indication that measurement of locomotor activitycould be usedas a methodto measure spatial mapping ability.The cpen-fleldtest maybe a usefulmethod to determine the functional statusof the hippocampus asa resultof drugtreatment, lesions,stimulation, etc.

Despitethe usefulness of theopen field test, moreelaboratetestingand recordingproceduresare necessaryto evaluateischemicdamageand more particularlythe effectiveness of therapyaimedat reducingtheconsequences I)f ischemic damage.Futurestudiesshouldutilizemultiplememorytests(e.g., radial armmaze); EEGrecordingand make the ischemicepisodessimilar to the clinicalsituation. Forexample,the occludingdeviceusedin Experimentt would be useful In establishing an animal modelresembling the clinical multi- episodestroke(Kato et al.,1989).The occlusionperiodcouldbe shortened

67

enoughtoresemble a transientischemiaattack(TIA). TIA resultin tempOrary amnesiaandmay occurmanytimesa day over a periodof severalweeks(Kato atal.,1989;Warlow and Morris,1982).Theoccluding devicewould be idealfor suchstudies.Pharmacologicalpreventionof ischemicbraindamagehas become oneof the most excitingareasin neuroscience.However,mostof the studies employ onlyhistologicalcriteriatoassess the effectiveness of these drugs.It perhapsismoreimportant to determineifneuronsthatappearnormal histologicallystill funclionnormally.The presentexperimentssuggestthatthe open-fieldlestwouldbea usefulfunctionalindexinconjunction with othertests to assessnewdrugsfortheirability topreventor reduce ischemicdamage.

68

REFERENCES

Agrawal,S.G.and Evans, A. H.(1986). The primaryafferentdepolarizing aclion of kainatein the rat.BfJ.Pharmacol 87: 345-355.

Alger,B.E.and Teyler,T.J.(1976). Long-term andshort-term plasticity in the CA1:CA3 and dentate regionsof the rat hippocampalslice.!llilin...Bn...110:

463-480.

Araki, H.,Nojlrl,M.,Kimura,M.andAihara,H.(1987).Effectof minaprineon 'Delayed neuronal death"In Mongoliangerbils with occludedcommon carotid arteries.J pharmacal Exp Ther 242: 686-691.

Auer,R.N"Jesen,M.L.,and Whishaw,I.Q.(1989).Neurobehavioral deficit due to ischemicbrain damage llrultedtohalf of CA1 sectorof the hippocampus.

J...t!oJIr2soi.9:'64'·'647.

Barnes. C.A.(1988). Spatial learningand memory process:the search for their neurobiological mechanismsin the rat.TrendsInNeufoscj.11:163-169.

Bederson,J.S.,Pitts,L.H.,Tsuji,M., Nishimura, M.C"Davis,R.L., Bartkowski.H. (1986). Rat middle cerebral artery occlusion:Evaluationof the model and development of a neurologicexamination.~17:472-476.

69

Benvenga. M.J'tWing,A.V.and DelVecchio,A.A.(1989).Effectsof NMDA antagonistson impairmentof the passiveavoidanceresponse in mice.

NeuroscienceAbstracts15:464.

Bliss,T.V.P,and Lorna,T.J.(1973).Long-lasting potentiationof synaptic transmissioninthe dentatearea ofthe anaesthetisedrabbitfollowing stimulatio n of thepertcrant path.~232,331·336.

Branthwaite,M.A.(1972).Neurological damage related toopen-heart surgery:Aclinicalsurvey.Ib.Q.rix.27:748.

Busto,H,,Dietrich,W.D.,Globus.M.Y.-T.,Valdes,I.,ScheInberg.P.and Ginsberg,M.D.(1987).Smalldifferences in intra-ischemicbrain temperature criticallydetermine the extentofischemicneuronal injury.JCOTeb. BloQdFlow Mo!ll!>.7:729-738.

Caronna.J.J.and Finklestein,S.(1978).Neurologicalsyndromesafter cardiac arrest.S1I2.M..9: 517.

Carpenter,M.B.and Sutln,J.(1983).Bloodsupplyotthe centralnervous system,inHuman Neuroanatomy (Carpenter,M.B.and Sulin,J.Eds.),Williams

&Wilkins,Baltimore.pp. 707-743.

Chandler;M. J .,Deleo,J.and Carney,J. M.(1985).Anunanesthetlzed gerbil modelof cerebral lschernla-lnducedbehav io ural changes.~

Moll!llll>.14:137-146.

70

Collingridge,G.L..Kahl.S.J.and McLennan, H.(1983).Excitatory amino acidsin synaptictransmission inthe SChaffer-commissuralpathway oftherat hippocampus.~334:33-46.

Collingridge,G.L.,Bliss,T.V.P.(1987).NMOAreceptors-theirroleIn long- termpotentiation.Trendsin Neyrosci.10: 288-293.

Cooper.A.(1836).Someexperimentsand observations on tyingthe carotid and vertebralarteries.Guy'sHasp.Rep 1: 457.

Corbell,D.• Evans,S.,Thomas,C.,Wang,D.and Jonas,R. A. (1990).MK·

801 reducescerebralIschemicInjurybyInducinghypothermia.~,514:

300-304 .

COlman,C. W.and Nadler,J. V.(1981).GlutamateandAspartate as hippocampal transmitters:biochemicalandpharmacologicalevidence. In Glutamate' Transmitterin The CenlralNervoys System(Roberts,P.J.,Storm- Mathisen,J.andJohnston,G.A. A. Eds.),. Wiley, Chichester,PP117·153.

Cotman,C.W.and Iversen,l.l.(1987).Excitatory aminoacids In the brain - focus on NMDAreceptors.Trends InNAuroSC;10:263·265.

Cotman,C.W"Monaghan,D.T.,Ottersen,O.P.and Storm-Mathisen,J.

(1987).Anatomicalorganizationof excitatory amino acid receptorsand their pathways.Trends in Neuroscl 10:273·280.