Permanent, 3-stage, 4-vessel occlusion as a model of chronic and progressive brain hypoperfusion in rats: a neurohistological and behavioral analysis

Behavioural Brain Research 160 (2005) 312–322

Research report

Permanent, 3-stage, 4-vessel occlusion as a model of chronic andprogressive brain hypoperfusion in rats: a neurohistological

and behavioral analysis

Carolina Justus Buhrer Ferreira Neto, Ricardo Alexandre Paganelli, Arcelio Benetoli,Keli Carina Miltus Lima, Humberto Milani∗

Department of Pharmacy and Pharmacology, Health Science Center, State University of Maring´a, Av. Colombo,5790, CEP 87020-900 Maring´a, Parana, Brazil

Received 13 September 2004; received in revised form 14 December 2004; accepted 17 December 2004Available online 20 January 2005

Abstract

Permanent, 3-stage, 4-vessel occlusion (4-VO) was evaluated as a practicable model of progressive, cerebral hypoperfusion in rats, resultingi anent andg e commonc rimentals fourg espitet . Morep .O .T t, althoughl nt,h able animalm©

K

1

t(lsig

mia,-

nent,del)ere-

-unc-are

uring,nent

0d

n quantifiable, reproducible, neuronal damage within a time interval shorter than that described in the 2-VO model. The effect of permraded 4-VO on cognition was also evaluated using the newly developed, aversive radial maze. The vertebral arteries (VA) plus tharotid arteries (CCA) or internal carotid arteries (ICA) were progressively and permanently occluded, following different expeequences (CCA→ VA; VA → CCA→ CCA or VA → ICA → ICA) with inter-stage intervals ranging from 1 to 4 weeks. Only two ofroups subjected to 2-stage 4-VO (CCA→ VA) showed modest reduction in the number of normal-appearing CA1 pyramidal cells, d

he significant treatment effect (p< 0.001–0.01 versus sham). A high rate of mortality (63.8%) was associated with 2-stage 4-VOronounced and consistent neuronal damage occurred 8 weeks after 3-stage 4-VO, following the sequence VA→ CCA → CCA (p< 0.001)ne month after this schedule, profound, persistent cognitive impairment was demonstrated in the aversive radial maze (p< 0.01–0.0001)his behavioral effect was not manifested when the ICA, rather than the CCA, were occluded, despite the presence of significan

ess severe, hippocampal lesioning. The mortality rate was significantly reduced when 3-stage 4-VO was used (p< 0.0001). These consisteistological and behavioral effects, combined with a low mortality rate, suggest that permanent, 3-stage 4-VO may represent a reliodel of chronic, progressive, cerebral hypoperfusion.2004 Elsevier B.V. All rights reserved.

eywords:Chronic cerebral hypoperfusion; Stepwise 3-stage 4-VO; Hippocampus; Neuronal damage; Learning and memory

. Introduction

Clinical and experimental evidence suggest a causal rela-ionship between the chronic reduction in cerebral blood flowCBF) and the development of dementia[6–8,12–14]. Patho-ogical conditions exist in humans, for example hyperten-ion, atherosclerosis, carotid artery stenosis and silent brainnfarcts, that may lead to chronic, progressive and severe re-ional cerebral hypoperfusion, mainly in aging patients who

∗ Corresponding author. Tel.: +55 44 2614814; fax: +55 44 2636231.E-mail address:[email protected] (H. Milani).

are subjected to major risk factors such as hyperlipidediabetes, smoking and others[7,15]. Chronic cerebral hypoperfusion can be provoked experimentally by permabilateral occlusion of the carotid arteries in rats (2-VO mo[10], which results in neuropathological changes of the cbral, microvascular endothelium[11,12], typical histologi-cal changes such as white matter rarefaction[30], and neurodegeneration and disruption of learning and memory ftion [16,21,29]. Such structural and functional changesthought to be reminiscent of the symptoms observed dhuman aging and dementia[7,8,11–13]. Characteristicallythe structural changes evolve very slowly after perma

166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2004.12.016

C.J.B.F. Neto et al. / Behavioural Brain Research 160 (2005) 312–322 313

2-VO, and the incidence of neuronal damage follows a non-uniform pattern. Generally, little or no neuronal loss or struc-tural damage to the hippocampal CA1 or other brain regionscan be observed before 3-month, permanent, 2-VO[7]. Themagnitude of brain damage varies widely among the differ-ent studies, as well as within the same group of experimentalanimals. For example, a 67% loss of CA1 cell was found4 months after permanent 2-VO in one study[16], whilein another only 2.6% CA1 pyramidal cells were damaged 7months after 2-VO[21]. Similarly, only sparse and sporadicCA1 hippocampal cell loss was observed 3 months after 2-VO in the rat[18]. In another study, there was no evidenceof hippocampal cell death even 6 months after permanent 2-VO [3]. Dark neurons, either isolated or grouped into smallfoci in the hippocampus were found both in CCA-occludedand sham-operated rats[27]. Tanaka et al.[29] reported onstriatal neurodegeneration 3 months after permanent 2-VO,but no quantitative data were provided, perhaps because ofthe large individual variability. These characteristics of the2-VO model may be explained by the fact that permanent2-VO in young individuals is not sufficiently effective in in-ducing sustained, uniform brain hypoperfusion, perhaps dueto the existence of a large and competent collateral bloodsupply[10], which may differ substantially among subjects.Further, young but not old rats exhibit an extraordinary abilityto auto-regulate cerebral blood flow after 2-VO[12].

d asa onic,n or re-v ss-i inlyd andm d re-q mager neu-r

erieso of ana esselo ni-f thisi uredum n usep -t

2

2

t thet tem-p at0 t thee

2.2. Surgery

Stepwise, 2- or 3-stage, permanent 4-VO was performed accord-ing to a modification of the method used to induce transient, global,forebrain ischemia[20,25]. Under ether anesthesia plus the localapplication of 2% xylocaine, the rats were subjected to occlusion ofthe common carotid arteries (CCA) and vertebral arteries (VA) fol-lowing the different combinations outlined below. In another groupof rats, the internal branches of the carotid arteries (ICA) were oc-cluded at a point posterior to the origin of the pterygopalatine arter-ies (PPA). The VA were permanently occluded by electrocautery. Alongitudinal incision (2 cm in length) was made into the dorsal neckto expose the alar foramina of the first cervical vertebra. The tip ofa unipolar electrode was inserted into the alar foramen and gentlyrotated until the presence of hemorrhage (1–2 s) assured that the VAwas ruptured. The hemorrhage was then stanched by electrocoagu-lation. The incision was sutured and the animal was returned to itshome cage. To ligate the carotid arteries (CCA or ICA), a similarincision was made into the ventral neck to expose the CCA, whichwas carefully dissected free from the vagus nerve and adjacent tis-sues. When necessary, a more distal dissection provided access tothe ICA/PPA bifurcation. Either the CCA or the ICA were perma-nently ligated by a suture thread, after which the incision was closedand the animal returned to its home cage. Animals assigned to thesham-operated group were subjected to the same surgical procedurewithout vessel occlusion.

2

2lows:

b n( iredi ectedt thec f theh 2-VOo trolg rimentw O oni n ofi

2the

s ls.T l oc-c ed toe logi-c ions.T im-i enceC ithi d/ori e pro-c ical,m reac-t thev wiseo

While the permanent 2-VO paradigm has been viewemodel to study some physiopathological aspects of chreurodegenerative disease affecting the human being (fiew, see references[6–8,11,12]), its usefulness in asseng the neuroprotective properties of drugs is limited, maue to the ample individual variability in the occurrenceagnitude of neurodegeneration. Further, the long periouired for the appearance of such sporadic neuronal daepresents an additional difficulty in the screening of theoprotective potential of drugs.

In the present study, we describe the results of a sf experiments performed to evaluate the developmentnimal model of progressive, permanent, 3-stage, 4-vcclusion (4-VO) with the objective of obtaining a more u

orm pattern of neuronal brain damage. The effects ofntervention on learning and memory were also meassing a novel version of the 8-arm, radial maze[20]. A per-anent, 2-stage, 4-vessel occlusion paradigm has beereviously by Plaschke et al.[22–24], although the histopa

ological outcome has not been reported.

. Material and methods

.1. Subjects

Male Wistar rats, weighing 280–300 g (70–80 days age) aime of surgery were used. They were housed at a controllederature (22± 1 ◦C) on a 12 h-alternate light/dark cycle (lights on7:00 h). Food and water were provided ad libitum throughouxperiments.

d

.3. Experimental design

.3.1. Experiment 1Rats were subjected to permanent, 2-stage 4-VO as fol

ilateral CCA ligation followed by bilateral VA coagulatioCCA→ VA). Different groups were prepared according the desnter-stage interval of 1, 2, 3 or 4 weeks, respectively. Rats subjo sham-operation or permanent CCA ligation (2-VO) formedontrol groups. The rats were killed for histological analysis oippocampus 1 week after the final stage of surgery (sham,r 4-VO), irrespectively of inter-stage interval. In the 2-VO conroup, the vertebral arteries were sham-coagulated. This expeas performed to evaluate the impact of chronic, 2-stage 4-V

ndividual survival rate and histological outcome as a functionter-stage intervals.

.3.2. Experiment 2Rats were subjected to graded, 3-stage 4-VO following

equence VA→ CCA→ CCA with 1-week inter-stage intervahese rats were then kept alive for 4 or 8 weeks after the finalusion stage or sham operation. This experiment was designvaluate the impact of 3-stage 4-VO on mortality rate and histoal outcome as a function of the duration and number of occlushe sequence VA→ CCA→ CCA was chosen because in a prel

nary trail (data not included here) we observed that the sequCA→ VA → VA profoundly stresses many of the animals, w

ncreasingly painful sensitivity and occasional inflammation annfection of the dorsal neck. This may have occurred because thedure requires two surgical interventions of the rigid, peri-cervusclo-skeletal structure of the dorsal neck. This traumatic

ion was avoided by firstly performing the bilateral occlusion ofertebral arteries through a single incision, followed by stepcclusion of the common carotid arteries.

314 C.J.B.F. Neto et al. / Behavioural Brain Research 160 (2005) 312–322

2.3.3. Experiment 3In a first group, rats were subjected to the occlusion sequence

VA → CCA→ CCA (Exp. 3a) with 1-week inter-stage intervals; 4weeks later acquisition performance was assessed in the aversive,8-arm radial maze. In a second group, the rats were subjected to thesame sequence of vessel occlusion, except that the internal branchesof carotid arteries were occluded (VA→ ICA → ICA) (Exp. 3b).The animals were divided into two subgroups and their behavioralperformance assessed 4 or 8 weeks after the 4-VO stage. Theseexperiments were performed to evaluate the influence of each 4-VOschedule on behavior and histology.

Table 1 illustrates schematically the experimental design de-scribe above.

2.4. Behavioral testing apparatus

We used the aversive, 8-arm, radial maze developed originally inour laboratory[2,20]. In the present study, we employed a confinedversion of the aversive radial maze, i.e., the animal was restricted tothe central area until release by the experimenter to explore the en-tire maze.Fig. 1provides a scheme of the confined, aversive radialmaze. In this device, eight arms (55 cm× 15 cm) radiate outwardfrom alternate sides of a central polygonal platform (71 cm across,16 sides). At the end of each arm, an opening (9 cm in diameter) pro-vides access to a darkened wooden box (23 cm× 11 cm× 9.5 cm),which can be inserted and removed like a drawer below any of theopenings, serving as a refuge for the rat (the goal box). Of the eighta s, theb ) bor-d l areai oors( ve thefl con-t r toc o ex-p alls, ac avail-a ated

constant noise in the testing room throughout the experiment. Twospotlights of 200 W each, plus a pair of ordinary incandescent lamps(40 W each) were fixed on the ceiling, 180 cm above the maze. Avideo camera was positioned 220 cm away from, and 130 cm above,the maze.

For descriptive data analysis, the eight arms were numbered ac-cording to their location in relation to the extra-maze cues such thatthe sequence and frequency of visits to each different location couldbe recorded.

2.5. Behavioral procedure

Four or eight weeks after permanent 4-VO, the rats were assessedfor behavioral performance. Before testing, the rats were habituatedto the testing apparatus. With free access to the arms, the rat wasplaced individually and directly into the center of the maze, and al-lowed to explore until it found the goal box, or until a 4-min periodhad elapsed. If the rat did not find the goal box within 4 min, it wasplaced into the arm containing the correct goal box into which itwas gently forced to enter by the experimenter. The rat was left for4 min within the goal box, and then returned to its home cage. Dur-ing habituation, the extra-maze cues were removed and the spatialposition of the goal box was randomly changed between subjects.This procedure was repeated for 3 days. On the next day after theend of habituation, training for acquisition of the task was carriedout for 15 consecutive days. The rats were trained using three tri-als/session, one session per day. For training, the rat was placed intot videoc peneds ntirem (con-t rmsw rea,t l wasa e ratf g thet thusf min.

TS manen ts ICA →

b tebral

rms, only one contained the true refuge; in the remaining armoxes were open-ended. Transparent, acrylic rails (2.5-cm highered each arm to prevent the animal from falling. The centra

s separated from the arms by transparent, acrylic guillotine d19 cm in height). The rotatable maze was elevated 90 cm abooor on a metal stand. From a separate room, a pulley systemrols each individual guillotine door allowing the experimenteonfine the animal within the central arena before releasing it tlore the arms. Several extra-maze cues (e.g., posters on the wlosed door, a window and some tridimensional objects) wereble in the room. A small ventilator located on the floor gener

able 1chematic representation of the experimental design for stepwise, perequences of vessel occlusions (CCA→ VA; VA → CCA→ CCA or VA →

CCAo, bilateral common carotid artery occlusion; bVAo, bilateral ver

he center of the arena, all arms now being closed, and theamera was turned on. Thirty seconds later, all arms were oimultaneously, and the animal was allowed to explore the eaze. When the rat entered half way down non-rewarded arm

aining a false goal box), the guillotine doors of the remaining aere lowered simultaneously. After returning to the central a

he newly-visited arm was closed immediately, and the animagain confined in the arena for a further 30-s period. When th

ound and entered half way down the rewarded arm (containinrue goal box), the guillotine doors of that arm were lowered,orcing the animal to enter the goal box, where it remained for 1

t, 2-stage 4-vessel occlusion or 3-stage, 4-vessel occlusion, according to the differenICA) used

artery occlusion; ICAo, internal carotid artery occlusion; w, week.


Fig. 1. Schematic representation of the confined version of the aversive, 8-arm radial maze. Each arm has a box just beneath the opening at the distal extremity;however, only one is the true goal box (closed box). Above the central area are two spotlights. The central area is separated from the arms by transparent, acrylicguillotine doors, which are operated from a separate room by a pulley system.

If the rat did not find the correct arm within 4 min, it was placed intoit and gently forced to enter the goal box. When the rat insertedonly its head into an incorrect opening and remained there for morethan 1 min, it was replaced at the center of the maze and the trialrestarted. If an animal persisted in this behavior for more than fourconsecutive sessions (days), it was excluded from the experiment.Between trials, the maze was cleaned of excrement, and randomlyrotated on its central axis; the goal box was randomly changed toanother arm, maintaining its spatial position unchanged in relationto the extra-maze cues.

The latency to find the goal box, the number of working mem-ory errors and the number of reference memory errors were usedto express behavioral performance. A reference memory error wasregistered when the rat visited an arm containing a false goal box(open-ended box) for the first time within a trial. However, if the ratreturned to an arm, which had been previously visited during thattrial, a working memory error was recorded. An arm was consideredvisited when the rat entered half way down its length. The animal

was considered to have left an arm when its entire body, includingthe tail, returned the central area of the maze.

2.6. Histology

At the end of each survival interval specified in Experiments1 and 2, or 1 day after the end of behavioral testing (Experiment3), the animals were deeply anesthetized, decapitated and the brainprocessed for histological assessment of CA1 pyramidal cell deathin the hippocampus. After decapitation, the brain was immediatelyremoved from the cranium and immersed in cold (1–2◦C) Bouin’sfixative. After 1 h, it was sectioned into two parts, which were im-mersed in the same fixative for a period of 3 days. Eight to twelve,paraffin-embedded, coronal sections (5-�m thickness) were takenfrom each brain at a level corresponding approximately to 4.52 mmposterior to bregma, and stained with celestine blue/acid fuchsin.Three coronal sections showing good quality staining were chosenfor bilateral counts of normal-appearing neurons in the dorsal por-


tion of the CA1 subfield. The number of intact-appearing pyramidalcells with a distinct nucleus and nucleolus, in each hemisphere, wascounted along a transect of 1.35 mm length (magnification 400×,field diameter: 450�m, Olympus). For each individual, the numberof pyramidal cells was expressed as the mean count in the threecoronal sections. The identity of the groups was not revealed duringhistological assessment.

The experimental procedures adopted throughout this study fol-lowed the “Basic Principles for Research Animal Use” set downby the Brazilian College of Animal Experimentation, and approvedby the Ethics Committee on Animal Experimentation of the StateUniversity of Maringa, Parana, Brazil (Protocol Number 022/2002-CEEA).

2.7. Data analysis

For each testing session (day), the behavioral performance cor-responding to the latency and number of errors incurred during theacquisition trials was calculated, for each rat, as the mean valueof three trials per session (day). Multifactorial analysis of variancefor repeated measures (MANOVA) was used to compare the per-formances across time, with ‘Groups’ as the ‘between’ subjectsand ‘Sessions’ as the ‘within’ subjects factors (Graph Pad PrismaVersion 4.00 for Windows). Where necessary, the Mann–WhitneyU-test was used to determine the time-points in which the groupswere statistically different. The Kruskal–Wallis analysis of vari-ance or the Mann–WhitneyU-test were used to quantify the degreeof CA1 pyramidal cell loss induced by permanent, 3-stage, 4-VO.A or-r lls int usingS ic 4-V siono ficantf

3

3 lc

ont d tob terw redt sub-j CA1p .T or 4-w CA1c thert eeki deaths risonw cute,g d re-c te,t era-

Fig. 2. Effect of permanent, 2-stage 4-VO (sequence CCA→ VA) on CA1pyramidal cell count as a function of inter-stage interval (left and belowabscissa; w, weeks). Histological analysis was performed 1 week after thelast stage. Intact-appearing cells were counted along a transect of 1.35 mmlength in each hemisphere. For each individual, the number of cells plottedrepresents the mean for three coronal brain sections. TCI, transient cerebralischemia induced by 15 min 4-VO (historical data).*p< 0.01;** p< 0.001;*** p< 0.0001, compared to the respective control groups for permanent, 2-stage 4-VO (sham/1w/sham) or acute TCI (sham) (n= 6–10).

tion (82%) was observed, in contrast to the mild reductionseen 7 days after permanent, 2-stage 4-VO (CCA→ VA). Itis important to note, however, that one individual exhibit-ing severe CA1 neuronal damage (68.8%) was present in thegroup subjected to 2-stage 4-VO with a 1-week inter-stageinterval (group 2VO/1w/4-VO).

Fig. 3shows the histological effect of permanent, 3-stage4-VO, according to the sequence VA→ CCA→ CCA, with1-week inter-stage intervals. Unfortunately, the brains of rats

Fig. 3. The effect of permanent, stepwise 4-VO according to the sequenceV ell,c staget tage ofo longa al, then ctions.*

proportion like t-test was used to quantify mortality data. Celation between the number of intact-appearing pyramidal cehe CA1 subfield and behavioral performance was estimatedpearman’s correlation test. Only animals subjected to chronO were included in the correlation analysis, since the incluf sham-operated subjects may bias results in favor of a signi

alse correlation[17].

. Results

.1. Effect of 2- or 3-stage, 4-VO on CA1 pyramidal celount

Fig. 2illustrates the effect of permanent, 2-stage 4-VOhe number of CA1 pyramidal cells. Rats were subjecteilateral CCA occlusion (2-VO) and 1, 2, 3 or 4 weeks laere submitted to bilateral VA occlusion (4-VO). Compa

o the sham-operated group, only two of four groupsected to 2-stage 4-VO exhibited a reduced number ofyramidal cells (K–W = 35.08, Dunn’s test:p< 0.001–0.01)hese were the groups that underwent occlusion at 1-eek inter-stage intervals. In these groups, however,ell density did not differ from that measured in the owo groups also subjected to 2-stage 4-VO, at 2- or 3-wnter-stage intervals, respectively. The modest neuronaleen in the first two groups can be emphasized by compaith the degree of CA1 cell loss measured after 15-min alobal, cerebral ischemia (4-VO model) in a previous anent study from our laboratory[2]. Seven days after acuransient forebrain ischemia, profound CA1 neurodegen

A → CCA→ CCA on the number of intact-appearing CA1 pyramidal compared to 2-VO and 3-VO. A period of 1 week elapsed from oneo the next, and damage was assessed 8 weeks (w) after the last scclusion (2-VO, 3-VO or 4-VO). Intact-appearing cells were counted atransect of 1.35 mm length in each hemisphere. For each individu

umber of cells plotted represents the mean for three coronal brain sep< 0.01 vs. sham (n= 8–11).


Fig. 4. Representative photomicrographs of coronal sections of the hippocampus (stereotaxic coordinate:−4.52 mm) of rats presented in theFig. 3. The letters‘a’ ‘b’ and ‘c’ indicate animals showing no, mild or severe CA1 cell loss, respectively, compared to the sham-operated group. Bar = 20�m (400×).

assigned to 4 weeks survival after the final occlusion stage(2-VO, 3-VO or 4-VO) were not available for histologicalanalysis, as they were accidentally damaged during theparaffin-embedding process. However, the mortality dataobserved in this group was used (see results below). After8 weeks, permanent, stepwise 4-VO caused consistentCA1 pyramidal cell loss in the hippocampus (18.6–62.5%;K–W = 14.91, p< 0.01 versus sham). There was no sig-nificant neuronal loss in the group subjected to 3-VO,despite the presence of neurodegeneration in two individuals(21.9–30.6%;p> 0.05 versus sham). CA1 pyramidal celldensity was not affected 8 weeks after 2-VO alone. Theindividual percentages of CA1 cell loss were calculated inrelation to the mean value for the sham-operated group.An animal was considered affected when its CA1 cellcount was less than the minimum value observed in thesham-operated group. Representative photomicrographs ofthe hippocampus of rats subjected to each of the various andsequential stages of vessel occlusion are illustrated inFig. 4.

3.2. Effect of stepwise vessel occlusion on cognition

3.2.1. Common carotid artery ligationFig. 5 shows the effect of stepwise, 3-stage 4-VO on

cognitive behavior assessed in the aversive, 8-arm radialm fol-l -s goalb orye er 4-V days( ncew ef-

fect on latency (F1,76= 10.0, p< 0.01), number of refer-ence memory errors (F1,76= 19.30,p< 0.001) and numberof working memory errors (F1,76= 24.89,p< 0.0001). Forall parameters, there was a significant ‘Group’ versus ‘Ses-sion’ interaction (F4,76= 4.47–11.32,p< 0.001–0.0001) anda significant, global ‘Session’ effect (F4,76= 4.34–15.72,p< 0.001–0.0001). However, when a one-way ANOVA wasapplied to each group individually, no Session effect wasseen in the hypoperfused group (latency, reference errors andworking errors:F4,30= 0.48–0.67,p> 0.05), indicating thatlearning performance did not improve across training. Underthis stepwise 4-VO condition, i.e., with CCA ligation, thenumber of intact-appearing CA1 pyramidal cells was signif-icantly reduced (panel D, Mann–WhitneyU-test,p< 0.001),ranging from 16.4 to 60.6% of the mean value for the sham-operated group. There was no correlation between the numberof intact-appearing CA1 pyramidal cells and acquisition per-formance, as measured by total latency, and number of errors(Spearman’s ‘R’ = 0.036–0.23,p> 0.05).

3.2.2. Internal carotid artery ligationIn this series, the rats were subjected to the same or-

der of vessel occlusion as described above, except thatthe internal branches of the carotid arteries were occluded(VA → ICA → ICA). Fig. 6 (panels A, B and C) showst thiss lysisb ef-f -s ters( p-e ntrolg d in

aze. In this experiment, progressive vessel occlusionowed the sequence VA→ CCA→ CCA, with 1-week intertage intervals. The mean latency to find the hiddenox, and the number of reference and working memrrors were measured during 15 consecutive days aftO (days 33–48), and are presented in blocks of 3

panels A, B and C, respectively). Acquisition performaas disrupted as revealed by a significant ‘Group’

hat acquisition performance was not affected aftertepwise 4-VO schedule, whether the behavioral anaegan 4 or 8 weeks after the 4-VO stage (‘Group’

ect: F2,96= 1.10–2.99,p> 0.05). A highly significant ‘Sesion’ effect was observed, however, for all parameF4,96= 14.47–33.29,p< 0.0001), indicating that the hyporfused group acquired the task at a rate similar to the coroup. The number of CA1 pyramidal cells was reduce


Fig. 5. The effect of chronic, stepwise 4-VO on acquisition performance and CA1 pyramidal cell density. The vertebral arteries (VA) and common carotidarteries (CCA) were occluded according to the sequence: VA→ CCA→ CCA. Behavioral testing began 4 weeks after the 4-VO stage (4-VO/4w) and extendedfor 18 consecutive days, including 3 days of habituation. For each individual, the mean value measured in three trials/session (day) was used to expressperformance in terms of latency, and number of errors. Hippocampal CA1 cells were counted 49 days after 4-VO. Behavioral values are the mean± S.E.M., inblocks of 3 days.*p< 0.001 vs. sham-operated group. Sham:n= 14; 4-VO:n= 7.

the group surviving for 4 weeks until behavioral testing be-gan, although this effect was not quantitatively significant(panel D: 7.5–22.5% cell loss,p> 0.05; histology on day 49after 4-VO). In the group surviving for 8 weeks until testing,however, a “mild-to-moderate” degree of CA1 cell death wasdetected, which ranged from 21.1 to 43.4% of the mean valuefor the sham-operated group (K–W = 8.87,p< 0.01; histologyon day 79 after 4-VO).

3.3. Mortality

Fig. 7shows the mortality rate and includes animals thatdied either spontaneously after each stage of vessel occlusion,or were killed for technical reasons. Of those rats subjectedto 2-stage 4-VO following the sequence CCA→ VA, 16.7%died within the first 48 h of 2-VO, and 47.1% after the 4-VO stage. Cumulatively, a 63.8% death rate occurred duringprogressive, 2-stage 4-VO. This mortality rated was signifi-cantly reduced when 3-stage 4-VO was used following the se-quence VA→ CCA→ CCA. After bilateral VA occlusion (2-

VO stage), only 4.4% of the rats died compared to 16.7% afterCCA occlusion performed in the previous group (p< 0.01).Continuing the sequence VA→ CCA→ CCA, 5.9% of therats died after the 3-VO stage, and 13.8% after comple-tion of the 4-VO stage (13.8% versus 47.1%;p< 0.0001).Cumulatively, 24.1% of the rats died during the entire se-quence VA→ CCA→ CCA, compared to 63.8% after thesequence CCA→ VA (24.1% versus 63.8%,p< 0.0001). Thecumulative mortality rate was even lower (18.2%) when theinternal carotid arteries were occluded following the se-quence VA→ ICA → ICA (p< 0.0001–0.05; compared toCCA→ VA). The mortality rate did not differ betweenthe sequences VA→ CCA→ CCA and VA→ ICA → ICA(24.1% versus 18.2%;p> 0.05).

4. Discussion

In this study, we evaluated the effects of permanent, step-wise occlusions of both vertebral and carotid arteries (4-


Fig. 6. The effect of chronic, stepwise 4-VO on acquisition performance and CA1 pyramidal cell density. The vertebral arteries (VA) and internal carotid arteries(ICA) were occluded according to the sequence VA→ ICA → ICA. Behavioral testing began either 4 or 8 weeks after the 4-VO stage (4-VO/4w and 4-VO/8wgroups) and extended for 18 consecutive days, including 3 days of habituation. For each individual, the mean value measured in three trials/session (day) wasused to express performance in terms of latency, number of reference memory errors, and number of working memory errors. Hippocampal CA1 cells werecounted 49 or 79 days after 4-VO, respectively. Behavioral values are the mean± S.E.M., in blocks of 3 days. Sham:n= 11; 4-VO/4w:n= 9; 4-VO/8w:n= 7.

VO), with a view to producing an animal model of chronic,gradual cerebral hypoperfusion providing consistent and re-producible neuronal lesions. Thus, the use of young, adultrats was considered appropriate to evaluate the feasibility ofthe permanent, 3-stage, 4-vessel occlusion paradigm. We areaware, however, that the use of an aged animal model is morerepresentative of the age-related, cerebrovascular, neurode-generative disorders that afflict man. Future studies shouldthus validate the permanent, 3-stage, 4-vessel occlusion pro-cedure in the aged rat.

The 2-VO model of cerebral hypoperfusion has beenused to reproduce certain neuropathological and behavioralchanges that are reminiscent of those observed in victimsof Alzheimer’s disease and/or vascular dementia[7,12]. In aretrospective analysis, it has been estimated that permanent2-VO in rats results in an early, dramatic reduction of regionalcerebral blood flow, reaching approximately 66% in the cor-tex and 48% in the hippocampus 2.5 h after 2-VO. One weekafter 2-VO, CBF can be restored to approximately 35% in the

cortex and 26% in the hippocampus, reaching around 81% re-covery in both structures 3 months after 2-VO[12]. This maypartly explain why even sporadic neuronal damage is rarelyobserved before a 3-months period after 2-VO[7], althoughthe occurrence of isolated, individual cases of severe CA1damage has been reported ([16]; the present study,Fig. 1).Alternatively, the occlusion of a single subclavian artery plusthe bilateral occlusion of the common carotid arteries (3-VOmodel) has been proposed with the aim of providing morerapid, reproducible and quantifiable, ischemic brain damage[4]. In this model, regional CBF is reduced to 62 and 85% inthe hippocampus and cortex, respectively, but returns to nor-mal 9 weeks after 3-VO in the young rat. In this state, 25% ofthe hippocampal, CA1 pyramidal cells were damaged after 3weeks of 3-VO. Nine weeks after 3-VO, however, neuronaldamage was absent or very reduced (5%) in the hippocam-pus[5]. These data reveal a time-dependent recovery fromneuronal damage, which may also occur after permanent 2-VO. Thus, the neuronal damage indicated by the presence


Fig. 7. Rate of mortality (%) as a function of the sequence of vessel occlu-sion and number of vessels occluded. The percentage of mortalities occurringduring the sequences VA→ CCA→ CCA or VA → ICA → ICA are com-pared to that of 2-stage 4-VO (CCA→ VA) after each stage of occlusion,respectively. CCA, common carotid arteries, ICA, internal carotid artery,VA, vertebral arteries.*p< 0.05,** p< 0.01,*** p< 0.0001.

of shrunken, dark (argyrophilic) or acidophilic neurons after2-VO or 3-VO[5] may be transitory, at least in the young rat.This has important implications if the effects of drugs thatprotect against neurodegeneration are to be assessed in suchmodels. Further, a high rate of mortality may be expected tooccur shortly after the imposition of sudden, permanent 3-VO. The profound reduction in cortical CBF (85%) observedsoon after sudden 3-VO[5] may be considered similar tothat observed after acute, transient, global forebrain ischemia[26]. Also, occlusion of the subclavian artery may constitute acomplicated surgical procedure, which can limit studies. Al-though more consistent and reproducible neuronal damagecan be produced in older subjects, the difficulty in workingwith aging animals may represent an additional and seriouslimitation for many laboratories. Owing to these limitations,perhaps, the permanent, 2-stage 4-VO model has been usedpreviously[22–24].

Our initial intention in the present study was to evaluate thehistological effects of 2-stage 4-VO, a paradigm developedand repeatedly used by Plaschke et al.[22–24], although weused a different sequence of vessel occlusions. Statisticallysignificant, hippocampal cell death was revealed 1 week after2-stage 4-VO in two of the four groups assessed. The othertwo groups were not affected by the 2-stage 4-VO procedure.The reduction in CA1 pyramidal cells was minimal, however,when compared to that observed after acute, transient 4-VO,a roupsr omt re de-t , ther 2-V ingt greeo le to

that caused by acute, transient 4-VO. A similar result wasreported 1 month after permanent 2-VO[16]. This profound,but sporadic neuronal damage may be partially explained byindividual differences in the extent of colateral blood sup-ply to the brain, and consequent difficulty in restoring CBF[10,12]. It is also possible that more ample neurodegenera-tion might occur as the duration of 2-stage 4-VO increases,but given the high rate of mortality observed after 2-stage4-VO (sequence CCA→ VA), we did not continue studieswith this model.

A longer survival time with a low mortality rate afterchronic, 2-stage 4-VO might be possible, however, if dif-ferent combinations of vessels occlusion is considered[24].Considerations on the rate of mortality constitute an impor-tant aspect, which may partially define the feasibility of agiven animal model. Our data show that the rate of mortalitycan be significantly reduced if: (a) the sequence of occlusionsbegins with the vertebral arteries, and (b) 3-stage rather than2-stage 4-VO is used, at least as described here. By combin-ing these factors, mortality rate was reduced from 63.8% after2-stage 4-VO to 24.1 or 18.2% when 3-stage 4-VO was usedwith occlusion of the CCA or ICA, respectively. Further, thisprocedure renders the entire process less traumatic as judgedby the general state of health of the animals. The rate ofmortality observed after 2-stage 4-VO in the present study(63.8%) is markedly higher than that reported by Plaschke eta par-t optedi ar-t byP rterya An-o ts. Int rom7 1.8t ndP tingt e ageo hus,i sionmc usion(

, neu-r hent tage4 le-s sion.N osi-t emi hp ot bec O or3 tedmm pre-

s used to induce global cerebral ischemia. Since the geceiving 2- or 3-week inter-stage intervals did not differ frhe sham-operated group, and since no differences weected among the four groups subjected to 2-stage 4-VOeduction in CA1 neurons in groups 2-VO/1w/4-VO andO/2w/4-VO may reflect a random variation. It is interest

o note, however, that one subject exhibited a high def hippocampal damage (68.8%), an effect comparab

l. [24], estimated at around 8%. This difference may beially explained by the sequence of vessel occlusions adn each study. In our experiment, both common carotideries were simultaneouly occluded, while in the studylaschke et al., crosswise occlusions of one carotid and the contralateral vertebral artery were performed.ther important variable may concern the age of the ra

he permanent 2-VO model, mortality rate is reduced f1.4 to 3.6% when the age of rats is increased from

o 9.2 months, respectively[16]. Both the present data alaschke’s findings agree with such results, further indica

hat the type and sequence of vessel occlusions, plus thf the rats, may influence the model synergistically. T

n terms of mortality, our present 3-stage, 4-vessel occluodel (VA→ ICA → ICA or VA → CCA→ CCA) may be

onsidered similar to Plaschke’s 2-stage, 4-vessel occlcrosswise CCA→ VA) model.

Permanent, 3-stage 4-VO causes clearer, consistentonal damage in the CA1 region of the hippocampus whe rats were allowed to survive for 8 weeks after 3-s-VO (31.8%;Fig. 3). The occurrence and magnitude ofioning depend on the number and type of vessel occluo CA1 lesioning was evident 8 weeks after the imp

ion of 2-VO; after 3-VO, only sparse cell loss was sen a few animals (Figs. 3 and 4). This finding agrees witrevious studies in that hippocampal cell damage cannonsistently observed before 12 or 9 weeks after 2-V-VO, respectively[5,7]. Other investigators have reporinimal [16,21]or no hippocampal damage[3] until 4 or 6onths had elapsed, respectively. Unfortunately, a more


cise analysis of the time course of neurohistological change(Fig. 3) was impaired in the present study, since the brainsof those rats surviving for 1 month after 3-stage 4-VO wereaccidentally lost. Thus, we do not know whether significantCA1 cell death might be present 30 days after the sequenceVA → CCA→ CCA. A uniforme and significant neurode-generation occurred, however, 47 days after the same pro-cedure (Fig. 5). For pharmacological reasons, however, it ismore important that such interventions provide a consistentand reproducible hippocampal lesion within a time less than2 months of graded, 3-stage 4-VO (compare the results ofFigs. 3, 5D and 6D). This is different from the 4- to 6-monthperiod required to observe minimal and sporadic, or evenabsence of CA1 lesioning after permanent 2-VO[3,21]. Theextent to which cerebral blood flow was permanently reducedcannot be estimated in the present study, but the severity ofneurodegeneration and behavioral changes may worsen as thechronicity of graded 4-VO increases. For technical reasons,the survival time after completion of the 4-VO stage could notbe extended in the present study. However, we are beginningto investigate the effects of longer periods of graded 4-VO(4–6 months), mainly by occlusion of the internal carotidarteries, and considering analyses of the visual system (seebelow for further discussion).

Another important aspect of the present study concernswith the effects of chronic, stepwise 4-VO on cognitive be-h thea r thec ar-t thes er ed byt Thisp de-s azet uredi thep ly anc ed ICAw ta reesw nent2 tionr -c ophyow rvediI beenr d forus theo only5 thse

considering that the radial maze task is highly dependent onvisual, extra-maze cues, disturbances of visual function mayhave contributed to the cognitive deficit seen after perma-nent, 3-stage 4-VO, using occlusion of the common carotidarteries (seeFig. 5). In another study, rats subjected to CCAligation (2-VO) for 12 weeks, and exhibiting important atro-phy of the optic nerves, were impaired in the radial maze task,despite the absence of morphological, hippocampal lesions[18]. Because of the possible influence of visual dysfunction,the role of hippocampal lesions in determining behavioral im-pairment cannot be established clearly after permanent CCAocclusion, despite the “mild-to-moderate” degree of CA1 cellloss observed in the present experiment (Fig. 5, panel D).However, changes other than CA1 cell loss also may con-tribute to the behavioral deficits. Disruption of complex be-haviors and recovery of function may reflect alterations at thesubcellular, synaptic or electrophysiological levels, or even ofwidespread morphological changes that cannot be quantifiedby a simple cell count in a restricted region of a given structure[1]. In fact, reduction of high energy, cerebral metabolism,measured in both hippocampus and cerebral cortex, corre-lates significantly with disruption of working memory in theholeboard test after permanent, 2-stage 4-VO[24].

In conclusion, the present study demonstrates that per-manent, 3-stage 4-VO is a practicable procedure that leads toconsistent and uniform, CA1 pyramidal cell loss, as well as top robe-h itiono hesec CCAo nedw tateo eo odelo t thei ow-e tem-a :( rseo ngers e ofc s ofv

A

anceb andU

R

. Anafter

avior. Permanent, stepwise 4-VO affected behavior inversive radial maze differently, depending on whetheommon carotid arteries (CCA) or the internal carotideries (ICA) were occluded. In the group subjected toequence VA→ CCA→ CCA, the capacity for learning thadial maze task was persistently impaired, as measurhe latency to find the goal box, and the number of errors.attern of cognitive impairment was very similar to thatcribed in rats with CCA occlusion and tested in the Y-mask[27]. Reference and working memory deficits measn the holeboard test, as well as memory disruption inassive avoidance task also have been observed acutehronically after 2-stage 4-VO[24]. In contrast, no cognitivisruption was observed in the present study when theere occluded (VA→ ICA → ICA). This differential effecccording to whether the CCA or ICA were occluded agith the findings of other investigators using the perma-VO model, and has been attributed to visual dysfuncather than to neuronal brain damage[3,18]. Permanent oclusion of the common carotid arteries may cause atrf the optic nerves and retinal degeneration in rats[3,18,28],hich is reminiscent of the blindness frequently obse

n human victims of occlusive carotid artery disease[9,19].ntense white matter rarefaction of the optic nerve haseported within 3 days of permanent 2-VO, and persistep to 1 month[30]. In the study by Ohta et al.[18], all ratsubjected to bilateral CCA ligation exhibited shrinkage ofptic nerves 3 months later. In another study, however,6% of rats subjected to bilateral CCA ligation for 3 monxhibited retinal and optic tract degeneration[3]. Thus, and

d

ersistent learning and memory impairment. These neuavioral changes occur within 2 months after the imposf stepwise 4-VO. The time course and magnitude of thanges appear to be highly dependent on whether ther ICA plus vertebral arteries (VA) were occluded. Combiith a low mortality rate and favorable, post-surgical sf health of the animals, the VA→ ICA → ICA sequencf vessel occlusion may represent a reliable animal mf chronic, progressive, cerebral hypoperfusion withou

nfluence of possible, peripheral (visual) disturbances. Hver, future studies are required to investigate more systically the impact of the VA→ ICA → ICA paradigm on1) the integrity of the visual system; (2) the time couf neurohistological and behavioral changes, using a lourvival time (4–6 months or more); (3) the time courserebral blood flow during and after the various stageessel occlusion; (4) aging rats.

cknowledgements

The authors gratefully acknowledge technical assisty Marcos A. Trombelli. Research supported by CNPqEM.

eferences

[1] Aronowski J, Samways E, Strong R, Rhoades HM, Grotta JCalternative method for the quantitation of neuronal damage


experimental middle cerebral artery occlusion in rats: analysis ofbehavioral deficits. J Cereb Blood Flow Metab 1996;16:705–13.

[2] Benetoli A, Paganelli RA, Giordani F, Lima KCM, Cestari Jr LA,Milani H. Effects of tacrolimus (FK506) on ischemia-induced braindamage and memory dysfunction in rats. Pharmacol Biochem Behav2004;77:607–15.

[3] Davidson CM, Pappas BA, Stevens WD, Fortin T, Bennett SAL.Chronic cerebral hypoperfusion: loss of papillary reflex, visual im-pairment and retinal neurodegeneration. Brain Res 2000;859:96–103.

[4] de la Torre JC, Fortin T. Partial or global rat brain ischemia: theSCOT model. Brain Res Bull 1991;26:365–72.

[5] de la Torre JC, Fortin T, Park GAS, Butler KS, Kozlowski P, PappasBA, de Socarraz H, Saunders JK, Richard MT. Chronic cerebrovas-cular insufficiency induces dementia-like deficits in aged rats. BrainRes 1992;582:186–95.

[6] de la Torre JC. Impaired brain microcirculation may triggerAlzheimer’s disease. Neurosci Biobehav Rev 1994;18(3):397–401.

[7] de la Torre JC. Critically attained threshold of cerebral hypoperfu-sion: the CATCH hypothesis of Alzheimer’s pathogenesis. NeurobiolAging 2000;21:331–42.

[8] de la Torre JC, Stefano GB. Evidence that Alzheimer’s disease isa microvascular disorder: the role of constitutive nitric oxide. BrainRes Rev 2000;34:119–36.

[9] Duggan J, Green WR. Ophtalmologic manifestations of carotid oc-clusive disease. Eye 1991;5:226–38.

[10] Eklof B, Siesjo BK. The effect of bilateral carotid ligation upon theblood flow and energy state of the rat brain. Acta Physiol Scand1972;86:155–65.

[11] Farkas E, De Vos RAI, Steur ENHJ, Luiten PGM. Are Alzheimer’sdisease, hypertension, and cerebrocapillary damage related? Neuro-biol Aging 2000;21:235–43.

[ ging

[ vas-

[ atho-52.

[ ebralbiol

[ gni-cedrain

[ brallants.

[18] Ohta H, Nishikawa H, Kimura H, Anayama H, Miyamoto M.Chronic cerebral hypoperfusion by permanent internal carotid lig-ation produces learning impairment without brain damage in rats.Neurosci 1997;79(4):1039–50.

[19] Osborne NN, Casson RJ, Wood JPM, Chidlow G, Graham M, Me-lena J. Retinal ischemia: mechanisms of damage and potential ther-apeutic strategies. Prog Retinal Eye Res 2004;23:91–147.

[20] Paganelli RA, Benetoli A, Lima KCM, Favero-Filho LA, MilaniH. A novel version of the 8-arm radial maze: effects of cerebralischemia on learning and memory. J Neurosci Methods 2004;132:9–18.

[21] Pappas BA, de la Torre JC, Davidson CM, Keyes MT, FortinT. Chronic reduction of cerebral blood flow in the adult rat:late-emerging CA1 cell loss and memory dysfunction. Brain Res1996;708:50–8.

[22] Plaschke K, Ranneberg T, Bauer J, Weigand M, Hoyer S, Bar-denheuer HJ. The effect of stepwise cerebral hypoperfusion onenergy metabolism and amyloid precursor protein (APP) in cere-bral cortex and hippocampal in the adult rat. Ann N Y Acad Sci1997;826:502–6.

[23] Plaschke K, Martin E, Bardenheuer HJ. Effect of propentofylineon hippocampal brain energy state and amyloid precursor proteinconcentration in a rat model of cerebral hypoperfusion. J NeuralTransm 1998;105:1065–77.

[24] Plaschke K, Yun S-W, Martin E, Hoyer S, Bardenheuer HJ. Interrela-tion between cerebral energy metabolism and behavior in a rat modelof permanent brain vessel occlusion. Brain Res 1999;830:320–9.

[25] Pulsinelli WA, Brierley JB. A new model of bilateral hemisphericischemia in the unanesthetized rat. Stroke 1979;10(3):267–72.

[26] Pulsinelli WA, Levy DE, Duffy TE. Regional cerebral blood flowand glucose metabolism following transient forebrain ischemia. Ann

[ entmonRes

[ men-lmol

[ me-the

erfu-

[ rahiteRes

12] Farkas E, Luiten PGM. Cerebral microvascular pathology in aand Alzheimer’s disease. Prog Neurobiol 2001;64:575–611.

13] Iadecola C, Gorelick PB. Converging pathogenic mechanisms incular and neurodegenerative dementia. Stroke 2003;34:335–7.

14] Kalaria RN. Small vessel disease and Alzheimer’s dementia: plogical considerations. Cerebrovasc Dis 2002;13(Suppl. 2):48–

15] Meyer JS, Rauch G, Rauch RA, Haque A. Risk factors for cerhypoperfusion, mild cognitive impairment, and dementia. NeuroAging 2000;21:161–9.

16] Ni J-W, Ohta H, Matsumoto K, Watanabe H. Progressive cotive impairment following chronic cerebral hypoperfusion induby permanent occlusion of bilateral carotid arteries in rats. BRes 1994;653:231–6.

17] Nunn J, Hodges H. Cognitive deficits induced by global cereischaemia: relationship to brain damage and reversal by transpBehav Brain Res 1994;65:1–31.

Neurol 1982;11(5):499–502.27] Sarti C, Pantoni L, Bartolini L, Inzitari D. Persistent impairm

of gait performances and working memory after bilateral comcarotid artery occlusion in the adult Wistar rat. Behav Brain2002;136:13–20.

28] Slakter JS, Spertus AD, Wissman SS, Henkind P. An experital model of carotid artery occlusive disease. Am J Ophta1984;97(2):168–72.

29] Tanaka K-I, Hori K, Tanaka NW, Nomura M, Ogawa N. FK506 aliorates the discrimination learning impairment due to preventingrarefaction of white matter induced by chronic cerebral hypopsion in rats. Brain Res 2001;906:184–9.

30] Wakita H, Tomimoto H, Akiguchi I, Matsuo A, Lin-XI J, IhaM, McGeer PL. Axonal damage and demyelination in the wmatter after chronic cerebral hypoperfusion in the rat. Brain2002;924(1):63–70.

Documents

Permanent, 3-stage, 4-vessel occlusion as a model of chronic and progressive brain hypoperfusion in rats: a neurohistological and behavioral analysis