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CEREBRAL ISCHEMIA, SPATIAL
MEMORY AND LOCOMOTOR ACTIVITY
by
© Dong Wang M. B.
A thesis submitted to the School of Graduate Studies
in partial fulfillmentof the requirements for the degreeof
Master ofScience
Divisionof Beslc Medical Sciences
Faculty ofMedicine
Memorial University of Newfoundland
September6 th, 1990
"~ \
St.John's Newfoundland
1"'1
The author has granted an itrevocable non.xcluslvellcence eJlowIng theNaIionaIUbmyof canada to reproduce, loan, cflSlribute or sellcopies of hislher thesisby eny meansandIneny formor format,maIdogthisthesisavailableto interested persons.' .
The author retains ownershipof the copyl1ghtin his/her thesis.': Neither the thesis norsubstantial extracts fromit may be printed oroth erwise reproduced without hislher permission.
l'suteur aaccorde unelicence il'l'\Wocab&e etnon exclusive pennettant i\ la Bi~uenationaleeu Canada de reprodulre, pritter.oLStribuer auvendredescopes de sa tMsede quelquemaniere et sous queIque formeque ce soit pour mettre des exempWresdece tte these a Ia disposition des persoonesjnteressees .
L'auteur0005e(Ve tapropnete du droitd'atJteurqui protegesa these . Nill! tMse oldesextraltssubstantiels de cene-ct ne doivent litrelmpcimes au autrarnent reproduits sans sonautorisation.
ISBN 0- 315-65320- 5
To My Mother, Father and Sisters.
ACKNOWLEDGEMENTS
I would like to express my thanks 10Dr. D. Corbell for providing me with the
opportunity to work on this project and for his encouragement . gu idance and
support through all stages of this research. I would also like to thank Dr.R.
Neuman for his wise council and encouragement, Dr. P.Moody-Corbett for her
support and helpful comments, and Ms. S. Evans for her technical support.
I am grateful for the financialsupport provided by the fellowship from the
School of Graduate Studies and the bursary from the Faculty 01Medicine.
without this support 11 would not have been possible for me to study here.
iv
ABSTRACT
Stroke is amongone of the leadingneurologicaldisorders in clinics. The
purpose of this studywas to investigate the functionaldeficitsof the
experimental -stroke- animal. In order to gel a better understanding of the real
mechanismsbehindthese deficits, a systemallc observationof the behavioural
and pathological changes associated with cerebral ischemiawas carriedout i,l
three experiments In Mongolian gerbils.
In EKperimenl 1, an animal model using delayed, repetinvecerebral Ische~ la
was used to determine the behavioural and neuropathological changes
following multi-episodes 01cerebral Ischemia.
In Experiment 2, animals were pre-exposedto the lestenvironmentbefore
ischemiaandan attempt WdSmade to determine whether the post-ischemic
hyperactivity reSUlted from a simple change in motor functionor a defICitin
spatialmapping ability.
In Experiment 3, the M8l1'opathOlogical andlocomotor activity changes
resulting fromdifferentcarotid arteryocclusionduratiort:l (5, 10 and 15 minutes)
were investigated. An attempt was made to determine whether the graded
ischemiaresulting from the different ischemic durations resulted in graded
increases In locomotor aC1lvity.
From these threeexperimentsit is concluded thaI thebasis for theincreased
locomotor activity followIngan episodeof cerebral ischemia is an alteration of
spatial learning abllily.
TABLE OF CONTENTS
ACKNOWLEOG EM ENT -- ------... . ------. -.-••--••-------.---... -. - . I I I
ABSTRACT ••-••- -••••--------••••••••-------- -- --.---.-- Iv
TABLE OF CONTENTS .. ------------------------------------------------ v
LIST OF FIGURES ------------------ --..... ------.-------- ----- - --- vII
LIST OF TABLES --------------------••••••------- ------------------------- vIII
LIST OF ABBREVIATIONS ------•••••••-----------------------.-----.-•• Ix
GENERA L INTRODUCTION ----- - -- --- ---- 1
1. Cerebral ischemia -- ---- ••••----.-•••••---.--•••- --••- ---- --... 1
2. Neuroanatomyof hippocampus .-- - ••---- ••••.•--.-- --.--••• 5
3. Excitatory amino acids . - -.---- - - ••- ••••••••- -- 6
4 . Functional studies ••••••••-•••••••- _._-_._ 12
EXPERIMENT ONE -- ----- - .-••••••---------- -- -- -------- -- --- 16
Introduction -•••••••••••••••••_ _._ ••_ _ _.. - 17
Methods - ••-- - - --•••--- .-.- ••- - -- --. 17
Hesults ••- - -••-. - - - _-.-•••---- --_ .._- - --- •••-- -- --. 25
Discussion ----- - .--- --.-••-- -- •••- ---- .-.---- - ••••- -•••• 28
EXPERIMENTTWO .-...-•••••--------- -- -- --- ---- .------------ 35
vi
Introduction-_.._- _.••.•.- •••-••••••••._ _._ .••..•_ ._••__..••-_.•_--· 36
Methods-- -- ....•.•...- ..•.-- ---.-.-- - ------.-.---.•....._-_••••- 36
Results --.--.--.--- ...•.•.----.•..--.- .•..- .----- •.••.••••-- .••.---- 39
Discussion -- ..- ..--- .....•.--.---.•...•.-- - .••- -----._- 47
EXPERIMEN T THR EE •••••.....•_••.•.•••.•....•.••.._.•....••••... 49
Introduction •.•.--.-.•.....•.--.--..... •.••.•.•.- ...--.- - - --.•....····--- ··· 50
Method ••_••_._••••••••••_._••••••_••_. _••••••_•••••_••_•••••_••••••_•• 50
Results _._._..••••••••••••••••-•.••••..... ..•._._ _.._ __ - 51
Discussion •••••_ •••...••••••_•••.•••.- ••••••.._--.-_..._._-_...- ----- . 56
GENERAL DISCUSSION ••••...•••..•••.••••••••••.•••••••••.•••.••• 51
REFERENCES •.•..•.•.•••..••••...•••..•.••••••••.•••••••••••••_..•······ 58
vii
LIST OF FIGURES
Figure 1: Schematic Drawing of The Hippocampus ••---- 7
FlQure2: Diagram of OCcluding Device- - - --- - _ 19
Agure 3: Diagram of Oct luding Table - - - - - - . -.- - -_.__ 21
Figure 4: Open Field Activity Following Repeated lschemla - --- 26
Figure 5: Photomicrographs of CA1 Areas ••••-.... _.... --••••-•••--•• 29
Figure 6: Degree of CAl Cell Loss •••- -.- .-.--- -••••••••- •••-.-.. ... . 31
Figure 7: Open Field Activity in Familiar and Novel Testing
Environments -- - ••----.- ••- .. - --- - ••- - _ _• 40
FlQure8: Photomicrographs of CAl Areas - --.--.-- _ .- 43
Figure 9: Degree of CA1 Cell Loss - - --- - 45
figure 10: Open Field Activityof Graded Ischemia ••-.--- - 52
Ftgurs11: Photomicrographs of CAl areas 57
FlQure12: Degree of CAl Cell loss --- .-- - - - - - - 59
FlQure13: Comparison Between A.B. and Ischemia MOdal - - 64
viii
LIST OF TABLES
Table1: Comparison01l ocomotor Activity - - ._ ._ - _.- 54
Ix
LIST OF ABBREVIATIONS
ANOVA _. Analysisof Variance
AMPA ••. . d-amino-:'3-hydroxy-S·melhyl-4-isoxazolepropionic acid
AP4 ._._. z-arrmc-e-pncsorcncberyrate
APS ••••• z-emlno-s-phcspnono-vatencacid
AP7 ••••• z-arranc-r-phcscnoncneptanctc acid
CGS ••••• cis-4·phosphonomethyl·2-piperidine carboxylic acid
CPP _._._. 3-(2-carboxyl-piperazin-4-yl)propyl-1-phosphonlcacid
EM ••••• Excitatory Amino acid
LTP ••••• Long-termpotentiation
MK·801- · (+)-5-melh yl-10,11-dihydro -5H-dibenzo(a ,d)cyclohepten-5, to-Imine
maleate
NMDA •• • N-melhyl-D-aspartic acid
MelD ••-. MicrocomputerImageDevices
PCP ••••• 1o(1-phenyl cyctohex yl)-p lperldlne
GENERAL INTRODUCTION
1.Cerebral Ischemia
Clinicalfeatures01stroke
The goalof the presentexperimentswasto investigatethe relationship
betweencerebral ischemia. hippocampalpathologyand functionaldeficits in an
animalmodel.Beforegetting10the animalmodelof cerebralIschemia, a brief
reviewof clinicalstrokeis presented.
Stroke was recogni::iedas a groupof neurological syndromescalled It
Zhongleng " in a traditional Chinese medicine book " Huang Oi Net Jing" (Canon
of Medicine) about 500 B. C. This book describes the neurological syndromes
causedby "the imbalanceof the blood In tho body as a resultof catchinga cold
wind", In Europe, SOranus of Esphesus(A.D. 98-138)observedthe "hemiplegic
paralysis"most!lften occurringin the elderlyduringwinter(Porken, 1974;Fields
andlemak , 1989).
Nowadays,strokeis one of the mostcommonlife-threatening
diseasesandis the third leadingcauseof death in the UnitedStatesafterheart
diseaseandcancer(Wolf et at, 1986). TheAmericanHeartAssociation (1963)
reportedthat there are about500,000strokevictimseachyear. Thetypesof
ischemicstrokecan be identifiedby differentclinicalfeatures. Datafromthe
populationof Rochester, Minnesota(Garrawayat aI.,1983),giveus an idea
aboutmortalityratesamongdjff~rent stroketypes. In this report,the average
annualincidenceratesper 100,000populationfor specificstroketypesfrom
1975-1979 are cerebral infarction (75). intr acerebral haemorrhage (13),
suba rachnoid haemorrhage (11), stroke of uncertain types (4), and all types
(103). 10 addition, many medical disorders can cause strokes . These include,
anoxicflSChemicdamage of the brain following: cardiac arrest (Caronna and
Flnklesteln. 1978); cardiac surgery (Branthwaite, 1 ~ /2) ; trauma and dissect ion
of cervco-cerebret arteries (Oragon et al.• 1981). From the above it can be seen
that stroke has a high incidence, hll.Jh mortality rate and many causal factors. In
the search lor treatments 01this complicated disease, many different "stroke"
models have been examined in t:,e last 150 years.
Models of Cerebral Ischemia :
1). COmpletely cutting the blood supply to the brain - this method Is
represented by deceplteucn. Brown-Sequard made the first observations of this
kind in 1858 . He observed changes ot respiratory movement in decapi tated
dogs (Weinberger et al..194O). Later Haymnn and Barrier reported that
automatic reflexes were gone after 12 minut rs 01cerebral ischemia
(Weinberger at al., 1940). All ot tile functional observations In the decap itated
anima ls were focused on the ischemic effects on the nerve reflexes. respiratory,
cardiac regulatory and vasomotor centres (Heymans et el., 1937).
2). General cjrculatory~. this method is represented by stopping the
general circulation pr ior to reviving the animals. D ifferent methods have been
used: chloroform intoxication and resuscitation by intra arterial lnjeclio n of
epinephrine and ca rdiac massage (Crile and Dolly, 1908); electrical shock to
produce ventricular fibrillati on and cardiac massage 10 restore Ihe normal
heart beat; and haemorrhage or asphyxia (Weinberger et al., 1940).
3). Occlu;;;ion of the arterial sl,!QQJy - Usually the carotid orvertebral arteriesor
both are occluded. This method was first introduced by CoOP~I' in 1836, in
which he ligated both carotid and vertebral arteries in thedog. Since then a
great deal of work has been doneusing this ischemicmodel inclUding
establishmentin other animals (Gildea and CObb, 1930).
In recent years, the studyof cerebral ischemiahas tendedto userodent
models (Ginsbergand Busto, 1989). However, unilateralor bilateral occ lus~on of
the carotid arteriesdoesnot produceischemicbrain damagein the rat due to an
efficientcollateral circulation (Payan et aI., 1965; Eklof and Siesjo, 1972). In
order 10producecerebral ischemiaIn Ihis an:mal, systemichypoxia or
hypotension is required(Smith at aI., 1984; Nordstrom and Rehncrona, 1977).
In 1979, Pulslnelll and Brierley Introduceda four-vessel occlusionmethodin the
rat by electrocauterizingthe vertebral arterythrough the alar foramina of the first
cervical vertebra and reversible clampingof both carotid arteries.
4). Focal ischemia- Allof the ischemic models mentionedaboveinvolveglobal
ischemia. Recently, a method for occludlnq the middle cerebral arteryhas been
Introduced in which the artery is occluded by a subtemporal craniectomy
(Tamura et aI.,1981; Shlgenoet al., 1985; Bederson et aI., 1986). This model
relatesmore directly to the regional ischemia commonly observedclinically.
The last two models mentionedabove have some advantagesin different
studies,but theyare not the mostideal model in functionalstudiesdue to
surgical complications:such as damageof the vertebral arteries in the four
vesselocclusion model and open-skull surgery in the focal ischemia model.
Recently a gerbilmodel (describedbelow) of cerebral ischemia has been used
quite productively in researchon stroke.
Mongolian gerbils , Mariones ungujculatus are rodents of the family Cricetidae .
They are widely distributed in the regions of Mo ngolia and northeastern parts of
China (Rich, 1968), In 1966, Levine and Payan observed that gerbil~~ were
susceptible to cerebra l ischemia produced by occ lusion of the carotid arteries
(two-vessel occlusion) . This Is because of the special cerebral-vascular
circulation In the gerbi l. Generally, the brain is supplied by two pairs of arteries ,
the internal carotid and the vertebral. The latter fuse into the basllar arteries at
the base of the brain. The internal carotid artery divides into posterior, middle,
and anterior cerebral arteries . The two anterior cerebra l arteries join rostral to
the optic chiasm. The basilar artery bifurcates into the two supe rior cerebellar
arteries. In humans and mosl mammals. there are a pair of arteries. the posterior
communicating arteries, which connect the posterior cereb ral and the basilar
artertea to form a "communicating" blood supply at the base of the brain called
"the circle of Willis" (Ca rpenter and Sutln. 1983; Vamori at aI., 1976). However.
there are no efficient connecting arteries between the carotid and the
vertebrobasilar arteries in the circle of Willis In gerbils (Levine and Sohn, 1969 ;
Kahn, 1972; Harrison et al., 1973). This makes it possible to produce foreb rai....
ischemia in gerb ilSby occluding the carotid arteries . The gerbil model has
features of simple surgery, fewer post·s urgical comp lications and effective
ischemic damage compared with some 01the ischemic models mentioned
before.
From the animal models of Ischemia it has bee n poss ible to classify the brain
damage observed into 2 types:
1). Selective neuronal vulnt:lrability. The most extensive brain damage
following ischemia is in the hippocampus, Specifically, neu rons in CA1 area are
most sensitive to ischemic insult (Ito at at, 1975 ; Kirino, 1982). Since the
present experiments focus on the post-ischemic functional deficits closely
related to the ful' io n of the hippocampus, a brief review 01the anatomy and
neurotransmitters of the hippocampus is presented below,
2) . Pan-necrosis. In this type the damage not only effects neurons but also
the glial and vascu lar ce lls. Prolonging the duration of ischemia or irreversible
occlusion of the blood supply will transform selective neuronal damage into
pan-necrosis (Hassman and Kleihues, 1973; Kirino and s er e. 1964), To study
more specific functional defic its which is only related to certain brain structures
this type of damage is avoided,
2. Neuroanatomy 01 Hippocampus
The hippocampus in mammals consists primarily of pyramidal neurons and
associated interneurons. These neurons are packed together in one layer of a
three-layered structure, compared to the six-layered structure in the cortex
(O'Keefe and Nadel, 1978). The hippocampus (Figure 1) Is divided into t~
major parts, the fascia dentata (dentate gyrus) and hippocampus proper (cornus
ammonis). The hippocampus proper has been further div ided into four subfields
Comus Ammonis (CA)1-4 (Lorente de No. 1934), CAl refers to the area "regio
superior" which is located near the distal end of the dense plexus. CA2 and CA3
are mainly included in the area called -regia inferio r" which are located at the
dentate end, eM stands for the pyramidal cells and interneurons scattered
inside the hilus of the fascia dentata. There are many hypotheses about
neurotransmission between the different regions within the hippocampus
(Frotscher et al, 1988). In order to get a clear view about this
neurctranamission . a conc ise description of the input,
transmission and output of the hippocampus (Zola-Morgan at at, 1966) is g iven
here. As shown in Figure 1, this neural circuitry finishes as a closed loop in the
entorhinal cortex. Among the pathways mentioned above, there are three
pathways using excitatory amL"'IO ack:ls (EM) : 1. The perforant pathw ay forms
the main glutamalelaspartate - containing aff~ents in the hippocampus
(Fonnum, 1984); 2. The mossy fibres terminating In the stratu m Iucid.Jm (White
at al., 1977); 3. The commissural fibres (contralatera l) and Schafter colfaterals
(ips ilateral) from CA3 which terminate in CAl and CA3 (Cotma n and Nadler,
1981).
Besides the glutamate sy stem there are many hypotheses about
neurotransmission in the hippocampus, e.g., cholinerg ic system and S·HT
system etc. ( Frotscher et al., 1968). Because it has been found that the
glutamate system has a dual role in memory and ischemic neu ronal death. the
role of the excitatory amino acid is emphasised here.
3. Excitatory amino acids
In the early 1960's, glutamate was rust proposed as a neuro excitatory agent
by Curtis and Watkins (1963) . Recently EM have been suggested to be
implicated in learning, memo ry and other brain functions. In add ition to their role
as neurotransmitters the EM are also neurotoxic at hIgh concentrations (Harris
et al., 1984; Clineschmldt et al., 1982; Schwarcz et al., 1984; Cotman and
Iversen, 1987; Rothman and Olney, 1987; Monaghan et al., 1989) . Within the
last 10 years it has become clear that there are multiple EM receptors, not just
the glutamate receptor as ori ginally thought.
Figure I. Schematic drawing of the hippocampus .
The lines showthe unidirectional pathway(adapted fromZola-Morgan crat. 1986).
(I ). Entorhinal cortex (EC) (major input)I (pcrforant pathway)
(2). Dentategyrus (OG)I (mossy fibers: excitatory)
(3). CA3I (Schatfercol latera ls)
(4). CAlI
(5). Subicular cortex(5)I
Entorhinal cortex.
Theexistenceof EM receptorsubtypeswasdeducedby the relativeactions
01selectiveagonistsand antagonists(CotmanandIversen, 1987). Based
primarilyon agonistresponsethe EM receptorshavebeenclassifiedinto five
types(Watkinset e..1990): 1) N-methyl-D-aspartate(NMOA); 2) o-amlno·3
hydroxy·5-melhyl· 4-isoxazolepropionic acid(AMPA); 3) kainate(K); 4) 2-amino·
4- phosphonobutyrate(AP4); 5) metabotroplc receptors.
NMDA, a glutamateanalogue,isa selectiveagonist whichhas
receivedconsiderable attenton asseveralfunctionshavebeen attributedto the
receptor activated by this agonist (seebelow). Radioligand autoradiography
showsthat the highestlevel of NMOAbindingsitesin the brain Is in the
hippocampus (Monaghanet at , 1963;Monaghanand Cotman,1985; Mayer
andWestbrook,1987). Within the hippocampus, the CA1region showsthe
highest levelof NMDAbindingSites, whereasmoderatelevelsare found inCA3
and dentategyrus(Geddeset al., 1986; Monaghan and Cotman.1986).
AMPA, a structuralanalogueofquisquallcacid, Is morespecificIn binding
studies thanqulsquatate(theoriginalagonist usedto define this receptor)
(Honoreet ai, 1982; Monaghanat at, 1989). The distributionof AMPAbinding
sites in the central nervoussystemis parallel to NMDAbindingsites. The
functionof theAMPAreceptorinvolvesgenerationof fast EPSPsInmanycentral
EM pathways(Monaghanet al. , 1989;Watkinsat at. 1990).
Kainate isnot as specific an agonistas NMDAandAMPA, and some
responseto kainatemay bemediatedby AMPAreceptors(Monaghan et ai"
1989 ; Honoreet aI, 1982) . However, the findingot a pure populationof kainate
receptorsoccurringon mammalianC-fibersIn the absenceof AMPAand NMDA
receptorsprovidesthe evidencefor discreteexistenceof kainate receptors
(AgrawalandEvans, 1986; Watkinset at , 1990).
10
The last two receptors, AP 4 and metabotropic are not as clearly understood as
th e former three recept ors, and work is still required to char acteriz e these
receptors (Monaghan at at . 1989; W alkins et at . 1990 ).
NMDA receptors and learning andmemory
One of the most exciting findings about EM is the role of the NMDA rece ptor
in theinduction of long-term potentiation (LlP) . l TP refers 10 a phenomenon in
whicha short burst0' high frequencystimulation results in polenliation of the
evokedpopulation spike. This potentiation may last for hours or days and is
wi dely reg arded as a cellu lar mode l 01learning and memory (Bl iss and Lorn a,
1973; Alger and Teyler , 1976; Collingrldge and Bliss, 198 7; Wroblewski and
Danysz.1 989).
The role of NMDA receptors in the induction of LTPwar; first demonstrated In
the CAl -eqion of hippocampal slice . LTP in the Sch affer/commissural pathway
is reversibly prevented by iontopho retic ad ministra tion of 2-amino-5
phosphono-valeric acid (A PS). a competitive NMDA antago nist . into the synapt ic
reg ion (Co llingridge et al.. 1983; H a rris et 81.• 1984 : Collingridge and
Bli ss,1987) . Also. phencyclid ine (PCP) and ketamlne . bot h non-competitlve
antago nists of the NMOA receptor p revent the induction 01 lTP in the CAl
region (Stringer and Guyen at, 1983). Morr is at al. (1986) found that chron ic
Intraventricular inject ion of AP5 co uld causa a se lective imp airment of place
learning in rats . and APS treatme n t also suppressed lTP in vivo. There are a
number o f other stud ies impl icating NMDA in learn ing and memory. For
example. other NMOA antagonists . e.g.• 3-(2-carboxy-,p ipe razin· 4· yl)propy 1-1·
ph osphonic acid (CPP). c is-4- phosphonome thyl -2. piper idine carboxylic ac id
11
(CGS19755)and PCP, havebeen foundto impair perlormance on passive
avoidance tests (Benvenga at at. 1989). (+)-5-methy'I-10,11-dihydro-5H
dibenz o(a .d)cycloheplen-S. 10- imine maleate (MK-801), a non-compe uve
NMDAreceptor antagonist, can produce impairmenton the radial arm maze
and increaselocomotor activity in the open field test (Shapiro and Caramanos,
1989; Heals and Harley, 1990). It seems NMDA plays an important role in
learningand memory. Unfortunatelythe same cetlularactionof NMDA receptors
which appear important for learning andmemory. can lead 10neuronal toxicity
when the receptoris excessively activated.
Ischemia inducedneuronal death
Many hypothes es have been put forth to explain the mechanism of neurona l
death following ischemic episodes . Neurotox icity 01excitatory amino acids
provides one of the most popular concepts about Ischemia induced neuronal
death, The neurotox icity of EM wa s first shown by Lucas and Newh ouse in
1957, who found tha t systemic inject ion of glutamate destroys the inner neural
layers of the immature mouse retina . Since th en many studies on the
neurotoxicit y ot the EAA have been carried out (Olney et at , 1971; Olney, 1978 ,
Monaghan et al., 1989). These stud ies show that systemic administration of
glutamata destroys neurons of the brain in newborn mice, rats or mon keys in
brain regions which lack a blood bra in barrier.
The select ive loss of neurons is seen in regions characterized by extensive
NMDA binding, and glutamate levels are increased in the hippocampus during
isc hemia (Jorgensen and Diemer, 1982; Cotman and Iversen, 1987). Some
EM antagonists can protect against ischemic brain damage. For example, local
12
administration 01a selective NMOA antagonist 2-amino-7-phosphonoheptanolc
acid (2-APH) into hippocampus effectively prevents Ischemic neuronal damage
(Simon et at, 1984); Systemic administration of other NMDA antagonists. e. g.,
MK·601, CG8-19755 andriluzolehave also beenreportedto protect against
Ischemic brain damage, giving furmer support to the proposed theory that
excessive NMOA activation may trigger ischemic brain damage (Gill 91at . 1987:
Margouris at at , 1989).
4. Functional Studies
From the above. it might be concluded that stroke is one of the most ilia
threateningdiseases. Tostudy this phenomenon many animal models of
cerebral ischemia havebeenestablished. In thesemodelsmost research has
focusedon thehippocampus because it was themost vuherable to ischemic
damage. Because the hippocampus plays an important role in learning an~
memory. it is important to understand the functionaldeficitsresulting from
hippocampal damage.
Scovillefirst reported a caseof severeanterograde amnesia followinga
bilateral medial temporal loberesection (damageto the anterior 2/3 of
hlppccampusandmedial temporal cortex)in a patient(H. M.) who sufferedfrom
intractable seizures In 1954. Although H. M. wasextensively studied (Scoville,
1954; Scoville and Milner, 1957) and continuesto be stooled. his damage W'J.S
clearly not confinedto the hippocampus. The conclusion that damage limite j 10
hippocampuscancauseamnesiawasnotcertain untilthecasereport of A. B.
by Zola-Morgan el al. in 1986. Patient A. B. sufferedseverelearning and
memoryimpairmentsfollowingan ischemic episoderesulting from coronary
f3
bypass surgery. Systematic memorytests performedduringthe liveyears
precedinghis deathshowedthat A.B. exhibitedsevereanterograde amnesia.
almost no retrogradeamnesiaandno significantchangein his personalityand
intelligence.Afterhe died,wholebrain sector-sweremade, and the most
prominent lesionsinvolvedthe CA1 regionof the hippocampus. Damageof the
CA1 region is theonly lesionthai could be found to explainR. 80'samnesia.
In animal studies,a varietyof teste havebeen useeto measurethe animals'
spatial learning and memory (Barnes, 1988). Many recent studies have been
performed to determine the relationship between functional defi cits (e.g "
learning and memory) and the hippocampus in ischemic models. An overview
of the tests which have been used in testlnq learning and memory in ischemic
models are described below :
1) . MQrrjs Water Maze. This test was first introduced by Morriset el. in 1982,
the basic principle of Ihe lest is thai there is an escape plattcrm which is hidden
beneath the surface of water, made opaque wilh milk, ;i1 a fixed location reialive
tc visible environmental cues outside th':l pool. Animals are released at random
ccations, and after a few trials the normal anima ls learn 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 10locate the platform (Morris at af., 1982).
Some studies have shown thai ischemic damage limited to half of the CAl
sector of the hippocampus can be severe enough to Impair the animals '
performance on this test (Auer et al., 1989). This finding gives further evidence
for an imponanl role of the CAl sector in place learning and memory.
2). Radial Arm Maze- With this experimental paradigm, animals obtain IQod
pellets in one of several arms, usually there are 810 10 anne. 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 referencememoryandworking memory.Reference memoryrelieson the
location of fixed externa l stimuli (e.g.• location of a window) relative 10each lest
arm. Thesecuesdo netchangefromtest day10testday. Working memoryrelers
to trial by trial changes in that the animal must remember which arms have been
entered on a particular trial. It has been shown thai animals with hippocampal
lesions take longer to localethe rightarm than controlanimals(Olton1978;
Barnes1988). Peelerand Smith(1990)showedthat5-7 minutebilateralcarotid
occlusionin gerbil whichproduced severe bilateralloss of CA1cellsled to
significantlymore re-enlriesin the radial armmaze.
3). Passive AyoldanceTest- Eventhoughthis test is not a specific spatial
memorytest,somestudieshaveshownthatgerbils withischemicdamageof
hippocampus showpoorperformance on thistest (Malgouris et at, 1989;
Tominagaand Ohnlshl,S. T., 1989).
Differentfrom all the abovelearning and memorytests, Chandler et al.
(1985) introduced openfieldtests in ischemicstudies.Chandleret erestudy
showedthatthe mostprominentbehavioural changefollowing 5 minutesof
forebrainischemiain the gerbil is a large increasein locomotor activity, evident
at l4 hrs postocclusion andgradually diminishing10normalafter about5 days.
Ina more systematic study,Gerhardt and Boast(1988)examined the
relationship betweenlocomotor activityanddegreeof ischemic damageof the
hippocampus Ingerbils. In their studyischemicdamageof the hippocampus
wasinducedby bilateralocclusion of both carotidarteriesfor 20 minutes.
Lo..:omotoractivitywas recorded onthe firstand fourthdaysafterccckrslcn
Histological assessmentrevealedneuronaldamagein the CAl areaof the
15
hippocampus. The meandistancethat theischemic animalstravelled (as a
Index of locomotor activity) duringDay 1 and Day 4 was approximately three
foldcompare j to the control animals. tI was found thatmere was a positive
correlation betweentheposlischemic hyperactivity and thedegreeof neuronal
degeneration in the hippocampus.
Comparingthe openfieldtest to the other threetests mentioned above. it
seems that open field lest hastheadvantage of simplicity. However, it hasnot
been established that the open field testis an effective funct'cnal test in cerebral
Ischemia experiments. Furthermore therehas not beena clear indication of the
mechanism underlying the post-ischemicincrease in locomotor activity. Isthe
Increase In locomotor activitya result of a simple motor hyperactivity or some
other runetional deficit. lor example, spatial learnIng or memory deficit ? In the
present experiments the relationship among cerebral ischemia, locomotor
activity and spatial memorywas studied.
16
EXPERIMENT ONE
17
INTRODUCTION
At present. most studiesof cerebral ischemiain gerbilsutilize a one-episode
Ischemiamodel. A few studies have involved repetitive cerebral ischemia in
which the intervalsbetween the episodesof cerebral ischemia were limitedto a
few hours (Tomida at aI., 1987, Vass et at, 1988. Kato at al., 1989). However,
these stud ies d id not examine any of the behavioural defic its resulting from
repetitive cerebral isc hemia. Results of a recen t study (Ge rhardt and Beast.
1988) suggest thai the degr ee of hippocampal damage is posltlvely correlated
with increased locomo tor activity. Concerning the relat ionship between severity
of the ischemia andthe degree of heightened locomotoractivation, would more
severe ischemicdamage, resulting from repeated Ischemicepisodesor longer
carotid occlusiontimes, produce corresponding IncreasesIn locomotor activity?
In the firstexperimant a model 01repeatedischemia (Tomida at at, 1987; Vass
et al., 1968) was used to address this question.
METHODS
SUbject s
Adult female gerbils were provided by High Oak Ranch Ltd, Goodwood,
Ontario, Canada, and weighed 45-70 g at the time of surgery. The gerbltswere
housed inplastic cages al a temperature of 22°C on a 12·hr day/night cycleand
fed with commerclal pellets and water ad libitum. All of the animals had been
resident In their cages for about four weeksbefore the experiments began.
18
Surgery
Animalswere anesthetizedby intraperitoneal injectionof sodium
pentobarbilal (50 mg/kg) followingpretreatmentwith an Intramuscularinjection
of atropine (0.1 1flg/kg). Theywere fixed in a supine positionand an Incision
about1.5 emin lengthwas made in the anterior cervical midline.Bothcarotid
arterieswerecarefully separatedfrom the surrounding tissue. A chronic
occludingdevicewas Implanted (Figure 2), suchthat the endsof the occluding
and releasing threads were directed subcutane ousl y 10the back of the neck
where they extended 1 em out of the skin . One week was allowed for the
animals tv recover from the surgery.
Prior to the occlusion, the animals were anesthetized In a plastic chamber
withether, andthe anesthesiawas maintainedby an -elher mask- (theopen
end of a syringewhich was filled with ether-soakedcotton). Occlusion was
accomplished by bilateral tightening of the occludingthreads and was
terminated by bilateral tighter;~.•l1of the releasingthreads (Figures2 and 3).
Animalswerekept under a 60 w lamp (SO em in height) from the beginning of
the surgeryuntil the animal recoveredfrom the anesthesia.
Procedure
Twenty-three adult female gerbilswere usedin this experiment.In the
ischemicgroup(n ::12), daily 10 minuteopen-field tests were begun?4 hrs
afterthe occlusionfor 4 successivedays In thefirst week. In the secondweek
the same proceduresof occlusion andopen field tests were performedagain.
19
Figure 2. Diagram of Occludi ng Device
The occludingdevice as described by Toroida et al. 1987.Theimplantation procedure wasmodified by threading theoccludingandreleas ing threads throug h the device with the aid of a loop of #6.0 silksuture.
20
~. .. L"""""
~~.:' ~"'''~
Lull (21
21
Figure 3. Diagram of Occluding Table
The occlusion wasmade by bil ateraltightening of the occludingthreads (Occ) maintained by two weights ( about 80 grams) hung at eacheachend of theoccluding threads. The occlusion was terminatedbybilatcrallight ening of the releasing threads (ReI).
22
ReI.
23
After finishing the secondweek of open field testing, four animals from this
group were perfused for histology. A third occlusion was performed on the
seven animals remaining in the group. These animalswere perfusedfour days
afteropen field testing.
In the control group(n =5). the sameprocedures of surgery were performed
but without occlusion. The open field tests were carried on for three weeks (four
test days per week).
A singleocclusion (10 minutes) wasperformed in six animals. The animals
wereperfusedfive days after the occlusion without behavioural testing. This
group was used10examine the histological changesafter a single ischemic
episode.
Open-tleld tests
Open-field tests wereperformed in a woodenbox measuring75em x 75em
x 50em. Thefloor wasdividedInto25 equal squaresof approximatly15 emx 15
em. The box was illuminated by two60 W lamps positionedabout 1.5 meters
abovethe floor. A viduocameramounted over the box wasused for recording
the activityof the animals. The number of squaresthe animalsenteredin 10
minutes wascounted as the measureof locomotor activity.
Histology
After the open field testingwas finished, the animalsweretranscardiajy
perfused with20 ml of 0.9% heparinized saline followed by 30 ml of 10%
buffered formalin. Thebrains were fixed in 10 % formalin at room temperature
24
ovemight. they were then cut into 40 urn coronal sections at ·23 0 C on a
freezing microtome. Sectionswere taken fromthe most rostral lip of the
hippocampus to 5.0 mm posterior 10 bregma. sect ions were dehydrat ed with
graded ethanols and stained with cresyl violet.
A Microcomputer Image Device(Me lD: Imaging Research Inc. Brock
University, St. Catharines. Ontario) was usedto assessdamageof the CAt
region of the hippocampus. The basic principle of MelD is to measure the area
of a brain structureusing the relative optical density. The relative optical density
of Ihe dentate gyrus waschosen as the targetvalue (similar 10normal CAt
relative optical density), andthe relative opticaldensityof the molecular layer
ventral to CA1 was chosen as the backgroundvalue. The thresholdvalue was
determined as follows:
ThreshOld value - (BV • TV)1O.4 + TV
BV-Background value
TV-Target value
The threshold value was crit ical in measuring the CA 1 area, because only the
relative optical den~ilY level above the threshold value could be detected . In this
experiment. the sections for MC ID analysis corresponded to a plane 1.6 mm
posterior to br8£ma . The CA1 region from its medial to lateral boundary was
delineated and the area exceeding the threshold value was measur ed. The
mean area (mm2) of both right and left hemispheres was combined to yield a
final score.
25
RESULTS
Gene ral obs ervations
Abnormalbehaviour wasnot evidentpost-lschemicajly, and the animals'
food and water intake appearednormal(noweight loss in post-lschemlc
animals), No adversesymptomswereobservedIn animalswith the chronic
occlusion device duringthe 3 weektestingperiod. An autopsyperformed on
one animalat the end of the third testing weekshOwed regenerative tissue
surroundingthe deviceandno infection wasobserved. Thedevicewas situated
in the original positionandworked perfectly.
Locomotor activity
As shown in Figure 4. locomotor activityof the ischemiagroup increased
after the first episodeof Ischemia IF(1,15)= 12.0, P<O.Ol, ANOVAj.and was
significantly elevated relative to the control group for the firs! three days (p <
0.01, Dunnett's Test). The second and the third occlusions lailed to Increase
locomoto r activity above contro l values (P > 0.05, Dunnett's Test). Locomotor
activity was maximal 24 hours after the first Ischemia, and was significant
compared 10the 2nd, 3rd and the lollowing test days (P<O.Ol, Dunnett's Test).
The level of locomotor activity of the control group remained relatively constant
over testing days while the locomotor activity of the ischemic group dropped
sharply, especially after the first two test days. This behavioural pattern yie lded a
significant Day effect (F (3, 45) "" 28.4, P < .00 11and a Treatment X Days
interaction [F (3,45) = 11.6, P < 0.01].
26
Figure 4. Open Field Activity Following RepeatedIschemia
Open field activityexpressedas squaresenteredper 10min testsession for theControl (C) and the Ischemia (I) animals. Days J..4represent the meanactivity scores.:tSEM for the I x 1 animals. Days5-8for the I x 2 animals and Days 9·12 for the I x 3 animals.
27
7001s t oeet ,
600
500
<>
'0e 400ScW..e 300•:><T1Il
200
100
00 2 3 4 5 6 7 8 9 101112
DAYS
28
Histology
Previous stud ies have shown the CAl secto r to be mainly respons ible tor
post- ischemic hyperactivityand spatial memory defICits( Gerhardt and Boast,
1988; Auer at at, 1989). Therefore. in this experimentand the other two
experiments . only the CAl sector was examined . It appears thaI CAl region
was progressively damaged alter each Ischemic episode (Figure 5).
Figure 6 illustrates the computer-generated measurement of CAl cell loss In
each of the experimental groups compared to the control animals (F (3,18) ""
5.B6, P < 0.01). From MelD results, some comparisons of C/\ 1 area (mm2)
were performed. eM areas in the two time (I )(2)and three time (I )(3)Ischemic
groups were severely damaged compared to the control group (Conlrol Vs. I x2:
p < 0.05; ControlVs. 1x3: p < 0.01; Dunnett's Test). Ho'ft'~er, CAt sector was
less damaged in the 1x I group, and did not reach statistical signir~f'Ir;A (P >
0.05, Dunnett's test). Therewas no statistically significant difference in damage
amongI x3 Vs. I x2 and I x2 v« I x1 (P > 0.05). Except I x3 v« I xt was
statisticaJty significant (FISher PLSD, P < 0.05).
DISCUSSION
In this experiment, locomotor activitywas transiently Increased after the first
ischemic insult, which Is consistent with earlier observations (Chandler et aI.,
1985; Gerhardt and Boast, 1988). However, additional ischemic damage
resulting from the second and third occlusions did not further increase the
activity level, which is not consistentwith the conclusion thai there is a positive
relationship between locomotor activity and CA1 damage (Gerhardt and Boast,
29
Figure S. Photomicrographs of CAl areas
Representivephotomicrographs takenfrom Control (A), 1x 1(8 ),1 x2(e) and I x 3 (D) animals. Note the progressive loss of pyramidal cells witheach subsequent occlusion ( B-D).
30
.. ' " ·f.. 0'
, ,-,
1DO AI m
31
Figure 6. Degree orCAI Cell Loss
CA l area (m m2) in the control and ischemic group s. Each barrepresents the mean area±5EM pooledfrombothhemispheres.
<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 presentexperimentand
GerhardtandBoast'sstudy is that thehippocampaldamageInthe present
experimentwas produced by repealedischemia episodes,and in their study
was producedafter a single occlusion.
Severalquestionswere raised bythis experiment:
1). Why did the first10 minuteocclusionnot result in significantCAl
damage, andwhy werethere somevariationsin the degree of damage of CAl
even in the same ischemicgroUfJ?
2). Why did locomotoraClivity increase only after the first ischem ic episode
and not after repeatedischemic episodeswhenhippocampaldamagewas
more extensive?
3). What Is the mechanism underlying these changes?
Topartlyanswerquestion 1. one has to go back to the methodsemployed.
This experimentwasdesigned beforeit wasappreciatedthat hypothermiahad
a protectiveeffect againstcerebral ischemia. Thus, in this experimentliltle
attention waspaid to body temperatureandthe "cooling effect" from the ether
anesthesia. This could account for the relativelyminor loss of CA1 aftera single
occlusionandthe variation in the degreeof damageof hippocampus, because
other experimentshaveshown that loweringthe bodytemperatureoneor two
degreescan allenuate ischemic damap,e (Bustoat al., 1987; Corbettel at .
1990).
For question2. hypothermiamightalso explain the significantdrop in
locomotoractivityover the first three post ischemicdays and the failureof the
secondandthird occlusion to Increaseit. The first periodof Ischemiacaused
incompletedamageof CA1 due to hypothermia. Thus,thq manyfunctioning
CA1 neurons[ef! after the first ischemic insultmay have allowedthe animals to
34
form a spa tial map of the op en field environment . This Is further supported by
the fact thaI the animals gradu ally dec reased their locomotor activity over the
first few post ischemic test days and dropped to the control level after four days.
Subsequent Ischemic episodes which causedmore severe ischemic damageof
the hippocampus would not be expected to aherthe locomotor activitybecause
the animars memory or map had already been formed (see General
Discussion ). The map ones formed is thought not 10 reside within the
hippocampus(Squire, 1986; 2ola-Morgan at at , 1986.).
The above results suggest that the increase In locomotor activity after
cerebralischemia(Chandler et al., 1985;Gerhardtand Boast, 1988; Present
results) may be due to some disruption of spatial mapp ing ab ility. In order to
further lest this hypo thesis . a second experiment was designed in which the
animals werepre-exposed to the test environment prior to the ischemia.
35
EXPERIMENT TWO
36
INTRODUCTION
As noted in thefirst experiment,repealed ischemicepisodesdid not further
Increaselocomotor activity, perhapsbecausethe animalhad formed a spatial
map of the test enviro nment If so, th en pre-exp osure to the open field prior to
Ischemic Insult should block or anenuete increased loc omotor activity since a
'spattal map" should have already been formed.
In the previousstudy itwas foundthat 10 minute occlusion with the occluding
device was insufficient inproducing effective CA1damage, so in this
experiment th e procedu re was modified in two ways: 1) In order to avo id
protec tive effect s of hypothermia all su rgical pr ocedures were carried out at
37.5 0 C; 2) Sinceonly a singleocclusion was to be performed,small
mlcrovescular clamps wereused instead of the polyethylene occluding device
that had beenusedin Experiment 1.
METHODS
Subjects
Fort y adult femalegerbils wererandomly dividedinto three groups: ischemia
(Group I. n =10). pre-exposure ischemia(Group FIN. n =10). pre-exposure
surgery control (Group FSN, n =10). andsurgerycontrol (Group S, n =10).
Surgery and occlusion
Anesthesia wasinducedIn a smallplastic chamberwitha mixtureof oxygen:
37
nitrogen: halothane at a flow rate 30% : 70% : 2.5%(mllrnin.). The anesthetic
was deliveredthrough a F1uot8C Anesthetic Machine (Foregger Co., Inc. Roslyn
Height, NewYorX. U. S. A). The animals were then fixed In a supine position
and halothane flowrate was reduced 10 1.5 %.
A two em anterior midline cervical incision was made. and both carotid
arteries were carefully separatedfrom the surrounding tissue and the
sympathetic nerves. Five minute occlusion 01the carotid arteries was made by
clamping the vessels with Schwartz mlcro-serreffnea (Fine Science Tools Inc.,
North Vancouver, B.C). After the occlusion the mlcro-serreflnes were removed
simultaneously from both carotid arteries. After making sure of good reflow in
both arteries, the animals' Incision was sutured and the animals were placed
under a 60 w lampIn theIr cages. The body temperature of the animals was
maintained at 37.5 0 C by a temperature.controlledblanket (HomeothemllC
COntrol Unit 482, Harvard Apparatus Ud, Edenbride, Kent, U. K) throughout
surger)'.
Open-fi eld test
Apparatus
Open-field tesllng was performed in the same lesting apparatus as in
Experiment 1. An image tracking system (VP112 scanning unit. HVS IMAGE,
Hampton, England) coupled to a Tatung·7000 computer was used to count the
number of squaresthat the animals entered during the testing period.
38
Te sting
InGroup I and Group S, daily 10 minute open-field teste were carriedout 24
hrs after occlusion/surgery andcontinuedfor 10 successive days.
In the Group FIN and Group FSN. the da ily 10minuteopen-field tests were
started fiv e days belor e the surge ry. M er five days of testing, the animals In the
FIN group were subjected 10a 5 minute bilateral occ lusion of the carotid
arte ries. For the FSNgroup, the carol1darte,;"s were Isolatedbut not occluded.
Open field testswere begun 24 hrs after occlusion/surgery andcontinued for
five days. After this five day test period, the activity of the pre-exposedgroups In
a seml-novel environment was exam ined . The test environment w a s altered by
inserting an elliptical wh ite plastic liner into the open field th ereby ob scuri ng the
white and natural woodwalls of the open field lest bOx. The floor w ascovered
with apieceof styrofoam. Only one light was used and its position relative to
the testapparatus waschanged. Pictures and other objects were hungon the
walls ct the lesting room , the cs iling was coveredby coloured picturesand the
vid eo camerawas wrappedIn a white coat. In addition, noise provided by a
large fan waspresent on novel test days. Animals of theFIN and FSNgroups
were then tested in this -never test environment lor fiveadditional days.
HIstology
Animals in the four groups were sacrificed about three weeksafte r occlusion.
Th e proceduresof perfusion and histological preparation and MelD analysis
were the sameas in Experiment 1.
39
RESULTS
Locomotor activ ity
The four experimental groups( Figure 7) consisted of two ischemia groups (I
and FIN groups) and two control groups (8 and FSNgroups).
As expected, 5 minutes of ischemia produced a large increasein locomotor
activityas illustrated by theperformance of Group I animals. locomotoractivity
of the I groupwashighly increased after the episodeof ischemiarelative to the
Sg roup I Atwo-factorANOVAwith repeated measures: F (1.16):: 75.1.P<
0.001). Locomotor activ ity of the I group anima ls dropped to a lower level by
thefifth day, but the level of locomotor activity was significantlyhigher than thai
of the 5 group even ten days after the occlusion (From Day 5 to Day 10, I Vs. S,
P < 0.0 1, Dunnett's Test}. The level of locomotor activity of the 5 group
remained relatively consta nt compared to the gradual reduction in locomoto r
activity of the I group (over the first five te sting devs, there was a significant
Group X Day interaction: F (9,1 44) =11.9, P < 0.001).
The locomotor activity of the I g roup gradually decreased over the first five
testing days, which resulted in a significa nt Day effect (F (9 . 144) =16.0 , P <
0.001] . Pre-exposure to the open-field pr ior to ischemia (Group FIN) virtually
blocked the post -ischem ic increase in locomotor activity. Although the I and FIN
groups underwen t the same ischemic insult, the locomolor activity of the I group
was sig nificantly higher than that of the FIN group in the fam iliar enviro nment
[F(1,16 ) .. 39 .6 , P < 0.00 1). Again there was a significant interaction (F(4,64 ) ""
6.2, P < 0,001] and significant Day effect [F(4,64) "" 15.2, P < 0,001) . Locomotor
activity of the I group was highly Increase d compared to that of the FIN group on
40
Figur e 7. Open Field Activity in Familiar and NovelTesting Environment
Daily mean j , Locomotor scoresof :
1).The ischemic animals ( Group I).
2).The surgerycontrol animals (Group S),
3).The pre-exposedischemic animals( Group FIN).( Familiar Environment +Ischemia + Novel Environment)
4).The pre-exposedsurgerycontrol animals ( Group FSN).(Fam iliar Environment + Sugery + Novel Environm ent)
41
1200
..,Cll
; 600cW
III2!co6- 400
l/)
o...
1000 NT
800 • 1r-8
-
200
2 3 4 5 6 7 8 9 10
DAYS
42
each of the five test days in the postlschemjc familiar environment(from Dayt 10
Day5. P < 0.01, Dunnett's Test).
Therewas nosignificantdifferencebetweenthe FINandFSNgroupsover the
first five test ingdays (F(1 .~6) =0 .3. P >O.5) even though the FIN gerbils had
beensubjectedto bUateralcarotid arteryocclusion. Thelocomotor activityof the
FIN group increased slightly over the first few days alter ischemiarelative to the
FSN group yielding a significant interaction (F(9,144) = 3.7, P<O.OOI]. The
locomotor activity in the novelenvironment was initially increased which
produceda significantDayeffect(F(9,144)=16.5. P <0.001).
in theFIN group,comparison of the locomotoractivityin thethree testing
periods(5 dayspre-ischemia, 5days post-ischemiain familiarenvironment. 5
dayspost-ischemia in novelenvironment) showed thattherewasno significant
difference amongthesethreeperiods[F(2. 27) = 0.5. n.s].
HistologV
Severe neuronalnecrosisandcell loss (Figure B)couldbe seen
in the CAl areaof hippocampus in bothI and FINgroups. TheCAl sectorwas
severelydamagedin bothischemic groups [I andFIN Vs. S andFSN;F (2, 27)
=20.8, P<.001J. Individualcomparisons betweengroupsshowedsignificant cell
lossin the I andFIN groupsrelativeto their respectivecontrols(P < 0.01,
ScheffeF~Test) . Therewasno difference in damage10CAl betweentheFIN
andI groups(Figure9),
43
Figure 8. Photomicrographs of CAl Areas
Representive photomicrographsofthe CA1areafrom a surgery control(groupS) animal (top) andfromanischemic ( groupI) animal ( bottom).Magnification 160 X. Cresyl violet stain.
44
100,u m
45
Figure 9. Degree of CA1 Cell Loss
Menuarea of CAl (mm2).±. S. E.M. in the control ( C ) and the ischemicgroups ( group I andgroupFIN).
'" P <0.01. Scheffc Test compared 10 control.
,...c(oc(WII:c(
0.3
0.2
0.1
46
•
• TT
0.0c F + I + N
47
DISCUSSION
In the present experiment, the I and FIN groups underwe nt the same
ischemic insult, however, the I group showed a significant increase in locomotor
activ ity white the FIN group showed activity levels com parable to the
cooperated control animals. Pre-exposure experience may thus have allowed
the animals to form a spatia l map of the enviro nment. Thus. for the FIN group
ischemicdamageof the hippocampus would not have lao much effect on the
locom otor activ ity because of the pre -acquire d spatial map. The hippocampus is
though t 10 be a statio n for info rmation-processing (Squ ire, 1986 ; Zo la-Mo rgan et
at . 1986) . The processedinformation (l.e.• a spatial map 01the open field)
wou ld be sto red in other brain structures, such as the cortex (Squ ire . 1976;
Zol a-Morgan et al., 1986).
On e would have expected the FIN group to show increased locomo tor
act ivity in the "novel" environment. However, there wa s no significa nt difference
among the activity sco res recorded d uring pre -ische mia, post-ischem ia and
"nov el" env ironment , which may be d ue to the ' never environment really being
familiar to the anima l. Althou gh , a number of stimuli in the testing room were
changed, a great many extern al cues (e.g., odour, lab shelves, video camera
etc .) remained constant the reby decreasing the M novelty" of the nove l
envi ronment.
O ne finding in this experimenllhat differed from previous studies (Ger hard t
and Boast , 1988) and from Experiment 1 was that the level of locomotor activ ity
of the I group never dropped to the co ntrol level , remaining significan tly higher
than that of the S group over all ten post-ischemic test days . Th is may be
explained by two facto rs in this experiment which are diffe rent from Expe riment
48
1: first , body temperature of the animals was maintained Ih rough-o ut the
occlud ing procedures. so thai there was no -hypot hermia- protection; the
second factormay be that the microvascular clips provided a more effective
occlusion of the carotidarteries than t:id the occludingdevice used in
Experiment 1. Thus, Ischemic damageof CAl was muchmore severein this
experiment than that in Experiment 1. In order to determine whether this
increase in locomotor activity was proport ional to the degr ee of ischemic
damage, Experiment3 was designed. In thisexperiment. a differing degreeof
damageto CAl wasproduced by varyingthe duration of occlusion.
49
EXPERIMENT THREE
50
INTRODUCTION
As mentioned in the general introduction, in Gerhardtand Boast's study
(1988) , the analysis of the different degree of ischemic damage of the
hippocampus was carried out In the same ischemic group (all Ischemic animals
underwent a 20 minute occlusion). Also in Experiment 1, the comparison of
hippocampa l damage was carried out using a model of ' repeated ischemic
episodes". In the presentexperiment, the relation betweenthe degreeof CA1
damage produced by varying the occlusion durationand locomotor activity was
exami ned.
The purposeof this experimentthen was10 seewhethermoresevere
ischemicdamageto CA1 would result in graded increasesin locomotor activity.
METHODS
aub tects
Thirteen gerbilswererandomly divided into two groups:10 minute occlusion
group (n :::: 8) and 15 minute occlusiongroup(n :::: 5). Thedata from S and I
groups of Experiment2 were addedto the datacollectedfrom this experiment.
Surgery and occlusion
The surgical and anesthetic procedureswere the sameas in the previous
experiment,except for the occlusion times (10 minutesand 15 minutes),
51
Ope n-fie ld lest
Daily 10 minute open-field tests were perf ormed fo r len days for each group .
The method of observation of locomotor activitywas the sameas in the previous
experiment.
Histo log y
The prep aration was the same as in the previous experiment.
RESULTS
Locomotor activ ity
As shown in Figu re 10 and Tabl e 1, Locomo tor activity of the ischem ia
groups increasedafter occlusion [F (3.29)=13. 8. P< 0.001]. Locomotoractivity
of the 5 minu te occl usion group (15) was sign ificantly increased compared to the
conlrol group IDunnet's Test: Day l -Day6, P <0.01; Day 7·Day 10. P < 0.05].
The locomotor activity of the 10 minute occlusi on group (110) and 15 m inute
occ lusion group (I 15) was significantly incre ased compare d to the contr ol group
from Day 1 to Oay 7 (P < 0.01) and Da y 6 (P < 0.05) and Irom Day 1 to Day 8
(P<O.Ol) respectively. Among Ihe ischemia groups locomotor activity of 115
group was increased significantly on 3 (Days 1, 4 and 5)01the nrst 5 lest days
[115 VS. 15 and110. P < 0.01). The locomotoractivity 01the ischemic groups
dropped sharply, especially for the 115 group (asignificantTreatment X Days
interaction:F(3. 9) ""43.0, P < 0.001). All four groups showeddailydecreases
52
Figure 10. Open Field Activity of Graded Ischemia
Ope n field activity expressed as squa res en tered per 10 minu te testsession.The dataof control and5 min ischemia groups is fromexperiment 2.
54
Table I. Comparison of locomotor activity
Control vs ischemic groups ( IS, 110. 115), comparedby Dunnett's Test.
15
110
115
Dayl --Day6
Dunnett's Test:
Day?
"
Day8 Day9
n.s
Day10
.... - P<O.OI• - -- P<O.Osn.s- nC'?significant
56
in locomotoractivity (asignificant Day effect: F (3, 27) =5.2, p " 0.001).
Hist olo gy
As shown in Figure 11 and 12, CAl necrosis can be seen in all ischemic
groups . A one way ANOVA showed significant CAl call loss IF (3 .29) "" 28.7,
P < 0.001). Subsequentcomparison by Dunnett's Test revealed significant CAl
cell [assIn each ischemicgroupcompared10control (p < 0 .01). However, there
was no clear diffe rence in severity of damage to CAl among the three ischemic
groups (P > 0.05) except 15 Vs. 11 5 (FisherPLSD. P < 0.05 ).
DISCUSSION
The post-ischemic increase in locomotor activity after 10 and 15 minute
occlusions was similar to that observedpreviously with a five minute occlusion.
The locomotor activity of the 115group was increasedsignificantly compared to
the 15 and 11 0 groups on Day 1, Day 4 and Day 5 but not on other test days,
which may be due to the small numberof the animals In the 110 (n ;::8) and 115
(n :::5) groups. This may reflect the somewhat greater CA1 damage sustained
by the 115group. However, it would seam thatchanges in locomotor activity are
near their maximal level after a five minuteperiod of Ischemia. The reason for
this "limited increase"of locomotor activitymay be due to "functional
specialization"within CA1, e.g., rostral half of CA1 sector. Thus more extensive
damage of CA1 sector will not have muchfurther effect on locomotor activity.
Another possibility Is that the level of locomotor activity Is near the maximal level
for normal exploratory behaviour, In other words, a behavioural ceiling effect.
57
Figure l l. Photomicrographs or CAI Areas
Representive photomicrographsof theCAI area fromalOminocclusionanimal (top) anda 15 min occlusion animal(botoro) .
Magnification 160 X. Cresyl violet stain.
58
1 0 0 urn
Figure 12. Degree of Ca ] Cell Loss
Mean area of CA l (mm2);t S. E. M. in the control (C ) and [he ischemicgroups ( 15. 110 and 115). T he data of control and 15 is from expe riment Z.
60
0 .3
0 .2
o Area CA1
<U~~.«
0.1
0 .0 -l-l-_--JL,...J.__.L.-JL..._.L.-L-_--'-
Contro l 1 5 110 I 15
61
GENERAL DISCUSSION
Extensive damage of the CAl region of the hippocampus was successful ly
achieved in the present studies. These results furthe r confirm the two-vesse l
occlusion in the ge rbil as an effective , convenient ischemia model for stroke
research (Levine and Sohn .• 1969: Kahn, 1972; Harriso n at al., 1973: Ginsberg
~nd Busto, 19B9 ). In Experiment 1, animals subjec ted to repeated ischemic
attacks did not show a graded increase In their locomoto r activ ity. This was
explained by the f9,clthal repeated ischemic episodes would not be expected to
change a preacquired spatial map formed after the first ischemic attack (not
seve re enough due to the protection by hypother mia) . In Experiment 2, pre
expos ure 10 th e open field prevented the post-ischemic hyperactivity activity.
This was a key obs ervation since it suggested that the increased loco motor
activity in the open field test was a spatial memory deficit rather than a motor
hyperactivity. In Experiment 3 , it was found that more severe ischemic damage
of CA1 produced by longer occ lusion durations (10 an c I') minutes) did not
increase locomotor activity substantially above levels produced by standard 5
minute occlusio n.
From all three exper iments it was found that there was a sensitive
relatio nship between the hippocampu s. spallal memory, ischemia and
locomotor activity. It should be interesting to see how these relate and affect
each other. First, one question that should be clarified is whether spatial
memory deficits rep resent a special functional deficit or a problem with general
memory? In order to answer this que stion, the brain structures involved in
spatial memory must be know n. Since different features of spatial analysis a re
j.
62
pertormed by different cortical systems , the information must be brought
together. The structu re wh ich encodes the relat ionship between the information
and the cortical system is thought to be the hppccampus (KOIband Whishaw ,
1990 ). Thus, patients with only hippocampal damage do not pass information
on to the cortical system , and should only show symptomsof topog raphical
disorientat ion rathe. than general memory impai rment (Zola-M organ et at .
1986; Kolb and Whishaw, 1990). This may welt be represented by the case of
H. M. His IQis aboveaverage (118 on the WechslerAdult Intelligence SCale).
He is quiet and well mannered socially. However, the following description is
evidence 01spattal deficits in H. M.••- After leaving the main highway, we asked
him for help in locating his house . He promptly and courteously indicated to us
severa l turns, unlit we arrive d at a street which he said was quite fa miliar to him.
At the same lime , he admined that we were not at the right address . A prone
call to his mother revea led that we were on the stree t where he used to llve
before his operation -- (Milner et at , 1968 ).
It used to be thought that H . M's spat ial problem resuned from his severe
memory problems because he h~·j comb ined lesions of the amyg dala and
hippocampus. However, as ment ioned above. H. M. has good memory of the
enviro nment wh ich he was familiar with before his surgery, and only shows
severe anterograde amnes ia. Th erefore, H, M.'s anterog rade amnesia is not
the result of a genera l memory problem. Recently, studies on A. B. showed that
ische mic damage to a small portion of hippocampu s, the CAl region , brought
on severe anterograde amnesi a (Zola-Morgan et al., 1986; Kolb and Whishaw,
1990).
In the present experj meo ts, animals did not increase locomotor activity in the
enviro nment which was familiar before the ischemic episo~e, but on ly showed
63
increased locomotoractivity in a completely novel test environment (e.g., group I
in Experiment 2) , which may be also called an "a nteroq rade topographical
amnesia". Comparingthis with the case of R.B(Figure 13), ltcan be seen that
amnesiaboth in R. B. and the ischemia modelhas a similarmechanism: CA1
loss, and theCAl losscancause anterograde amnesiaor anterograde
topographical amnesia.
So far it is clear that ischemic damage 01CA1 can cause anterograde
topographical amnesia. In the presentexperiments, if locomotor activityis taken
as an Indexof spatialmemory. it is necessaryto knowhow spatial memoryand
locomotoractivityare related. In addition, it isalsonecessary to discussthe
choice of the open-lield test as opposed to a radial arm maze or 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 mixture of different activities, for example, walking, drinking, eating,
sniffing, sleeping, grooming, etc. Eachspecies of animal engages in a certain
mixture of activities which make them differ from other species, and the
reactions to particular external stimulation mayvary from species to species.
However, there are certain patterns of behaviour that remain constant across
species. For example, in the situation of 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. Novelty elicits a state of anxiety,
and exploration is motivated by this slate . Spatial information obtained during
exploration will reduce fear and habituation to Ihe external stimulation will
develop (Halliday, 1968; Groves and Thompson, 1970). In theopen-field test,
64
Figure 13. Compar ison of R. B and Ischemia Model
It canbe seen that both anterograde amnesia in R. B. and anterogradetopographical amnesiainischemiamodel arecausedby the samemechanism : CA 1 loss.
65
66
the environment is novelto theanimal.The animalexplores the environment
and exhibits increased locomotor activity until a spat ial map is formed
(habituation). Threat, stress,food. wateranda sexualmatearethoughtto bein
the sameclassof externalstimuli(O'KeefeandNadel.1978). Animalstestedin
an open-tleld lest (stress of novel environment), Mor ris water 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 should be considered as testing an
aspect 01spatial memory, and post-ischemic changes of locomotor activity
could beused as a sensitiveindex to examine the animal's spatialmemory.
Theadvantagesof the open-fieldtestare its simplicity, easeof observation
and analysiscompared to thewater maze.Moreover,the animalsdonot haveto
be tooddeprivedas in the radialarm mazetest. It alsoallowsearl) testing after
the
ischemic insult. Previousopen-tteldstudieshave notgivena clear indication
that measurement of locomotor activitycouldbe 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 fieldtest, moreelaboratetestingand
recordingproceduresarenecessaryto evaluateischemicdamageandmore
particularlythe effectiveness of therapyaimedat reducingtheconsequences I)f
ischemicdamage. Futurestudiesshould utilizemultiplememorytests(e.g.,
radial armmaze); EEGrecordingandmaketheischemicepisodessimilar to
the clinicalsituation. Forexample,the occludingdeviceusedin Experimentt
would beuseful Inestablishing an animalmodelresembling the clinical multi
episodestroke(Katoetal., 1989).Theocclusionperiodcould be shortened
67
enoughto resemble a transientischemiaattack(TIA). TIAresultin tempOrary
amnesiaandmay occur many timesa day overa periodof severalweeks(Kato
at al., 1989 ;Warlow and Morris, 1982) . The occluding device would be ideal for
such studies. Pharmacological prevention of ischemicbrain damagehas
become oneof the mostexcitingareasin neuroscience.However, mostof the
studies employ only histological criteria to assess the effectiveness of these
drugs. It perhaps ismoreimportant to determineifneuronsthatappearnormal
histologically still funclion normally. The present experiments suggestthat the
open-field lest would be a useful functional index in conjunction with other tests
to assess newdrugsfor their ability to preventor reduce ischemicdamage.
68
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