Effect of intravenous administration of dipyridamole in a rat model of chronic cerebral ischemia

Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: Innate Inflammation and Stroke

Effect of intravenous administration of dipyridamolein a rat model of chronic cerebral ischemia

Alessia Melani, Sara Cipriani, Francesca Corti, and Felicita PedataDepartment of Pharmacology, University of Florence, Florence, Italy

Address for correspondence: Felicita Pedata, Department of Pharmacology, University of Florence, Viale Pieraccini 6, 50139Florence, Italy. [email protected]

Pharmacological therapy able to improve the cognitive performances of patients with chronic vascular pathologiescurrently remains unavailable. Many studies of chronic cerebral hypotension in rodents have revealed alterations inreference memory and learning. Dipyridamole was introduced into clinical medicine in the early 1960s as a coronaryvasodilator. It is a potent inhibitor of platelet activation and reduces formation of thrombi in vivo. In addition, it is anantithrombotic agent used for secondary stroke prevention in combination with aspirin. Recent evidence indicatesthat dipyridamole has anti-inflammatory properties. Bilateral common carotid artery occlusion (2VO) in the rat isrecognized as a valid model of chronic cerebral hypotension, also defined as the “vascular cognitive impairment ratmodel.” Here, we report that dipyridamole reverses the impairment of spatial working memory 90 days after 2VO.This protective effect might be in relation to dipyridamole’s anti-inflammatory properties.

Keywords: dipyridamole; chronic ischemia; two-vessel occlusion; cognition tests; neurological deficit

Introduction

Chronic ischemia is a progressive, dynamic pro-cess caused by cerebral hypoperfusion (CH) thatmay manifest with cognitive dysfunction as ischemicconditions persist and ultimately leads to neuronaldeath.1 Chronic hypoperfusion of the brain sec-ondary to vascular pathology may be a risk factorfor neurodegenerative diseases.2,3 Vascular demen-tia and Alzheimer’s disease are in fact frequentlyassociated with a common pathophysiological stateof chronic CH.4 Age-related cerebrovascular steno-sis caused by arteriosclerosis induces failure of thecerebral circulation. Resolution of CH such as thoseinduced by arteriovenous malformations or carotidstenosis/occlusion has been reported to improvemental decline in humans.5

Up to now there is no available pharmacologicaltherapy able to improve cognitive performance inpatients with chronic vascular pathologies. Thus, amajor goal in experimental research is to identifyand evaluate cognitive dysfunction using differentexperimental animal models to help elucidate thepathophysiological mechanisms that link CH withalterations in brain function.

Chronic CH in rats

Many studies of chronic cerebral ischemia orchronic cerebral hypotension have revealed alter-ations in reference memory and learning in ro-dents.6–12 Cognitive impairment, white matter le-sion, and microglial activation can be produced inrats by permanently clipping the common carotidarteries (two vessel occlusion: 2VO),13,14 with thehippocampus being the brain region that under-goes the most damage. The neurodegeneration inthe hippocampus, evaluated by hematoxilin–eosinor cresyl violet or TUNEL labeling, gradually pro-gresses with time from CH induction.15 Severalstudies of rats have demonstrated that hypoper-fusion can lead to behavioral impairment beforeneuronal damage is seen, suggesting that hypoper-fusion, without the presence of cellular damage, canproduce learning and memory deficit.9–11

Capillary diameter and vascular endothelialgrowth factor (VEGF) expression increase progres-sively after 2VO, suggesting that mechanisms tocompensate for chronic insufficiency in blood flowinclude vasodilation, VEGF expression, and neovas-cularization in the ischemic region.3

doi: 10.1111/j.1749-6632.2010.05732.xAnn. N.Y. Acad. Sci. 1207 (2010) 89–96 c© 2010 New York Academy of Sciences. 89

Effect of dipyridamole on spatial memory Melani et al.

Several pharmacological effects have been studiedin the 2VO model, aiming to preserve brain antioxi-dant capacity,4,16 reduce white matter damage,17 re-duce reactivity of white matter damage-associatedproteins,13 and reduce microglial activation.14

Cerebrovascular white matter lesions and mi-croglial activation are frequently observed in vas-cular cognitive impairment and vascular dementia.In older people, chronic hypoperfusion of the brainsecondary to vascular pathology may be associatedwith inflammatory events and oxidative stress. Sucha pathological condition may contribute to impair-ment of cognitive functions and memory, whichcharacterize old age.18

After 2VO, the COX-2 and eNOS enzyme lev-els are increased in both the hippocampus andfrontal cortex after 1 week19 and nuclear factor-kappa B (NF-�B), IL-1�, and TNF-� are upreg-ulated from 4 weeks after occlusion.20 Astrogliosisand microglial activation are already observed 3 daysafter 2VO with a peak of microglial activation at 7days and of astrogliosis at 14 days.21 The persistenceof microglial activation22 and astrogliosis23 up to3 months after 2VO have been described. HoweverFarkas et al.22 reported no astrogliosis 3 monthsafter 2VO. Increased oxidative stress has also beendescribed: lipid peroxidation was detected 3 daysafter 2VO,24 increased activity of malondialdehyde(MDA)25 and superoxide dismutase, and reducedactivity of glutathione peroxidase and catalase weredetected about 2 months after 2VO.16,25

Given above, chronic CH in the rat induced by2VO is recognized as a valid model of chronic cere-bral hypotension;12 it is assumed that it mimics, al-though with some limitations, a situation of chronichypoperfusion-induced human dementia.15 Thismodel is also defined as the “vascular cognitive im-pairment rat model.”20 After 2VO, changes in cere-bral blood flow can be divided into three phases:an acute phase immediately after the start of occlu-sion that lasts for a maximum of 2–3 days, a chronicphase of hypoperfusion that lasts from 8 weeks to 3months, and a restitution phase when the cerebralblood flow returns to baseline via compensatory andadaptive mechanisms.15 Although this model doesnot mimic the compromised cerebral circulationin aging or demented humans, it may provide anaccurate evaluation of neurological functions andcognition up to 3 months after occlusion. Recently,an attempt has been made to refine the 2VO model

to avoid the acute phase directly after occlusion, soas to examine neuronal consequences of graduallydeveloping chronic CH as occur in human agingand dementia.15 The model of occlusion of the twocommon carotid arteries separately, at an intervalof 1 week, would allow hypoperfusion to developmore gradually.12

Dipyridamole as an agent for secondarystroke prevention

Dipyridamole was introduced into clinical practicein the early 1960s as a coronary vasodilator. More-over, it was seen that chronic dipyridamole therapywas cardioprotective against ischemia-reperfusioninjury in guinea pigs, an adenosine A1 receptor-mediated effect26 and limited ischemia-reperfusioninjury in forearm skeletal muscle in humans,27 aneffect likely mediated by adenosine. Dipyridamole isa potent inhibitor of the adenosine transporter, thusincreasing extracellular adenosine concentration.28

By stimulating A2 receptors, adenosine is a potentregulator of blood flow29 and is involved in regu-lation of cerebral blood flow during hypotension.30

In addition, dipyridamole is a potent inhibitor ofplatelet activation31,32 and reduces thrombi forma-tion in vivo.33 These investigations have led to theuse of dipyridamole as an antithrombotic agent forsecondary stroke prevention34 in combination withaspirin. At that time, clinical trials on stroke pre-vention involving both dipyridamole and aspirinshowed that combination therapy was not more ef-fective than aspirin alone.35,36 The AICLA study35

and the American–Canadian Co-Operative StudyGroup on Cerebral Ischemia36 probably failed be-cause of the dipyridamole formulation used and theinsufficient number of patients included in the stud-ies. In fact, orally administered, “instant-release”dipyridamole probably did not reach pharmacolog-ically active plasma levels. On the contrary, recentresults of the European/Australian Stroke Preven-tion in Reversible Ischemia Trial (ESPRIT) clin-ical trial37 showed that the combination therapyof dipyridamole orally administered as “extended-release” plus aspirin is substantially more effectivethan aspirin alone in the prevention of secondarystroke. These results are consistent with those of an-other clinical trial (ESPS 2, Second European StrokePrevention Study)38 that also showed a benefit ofthe same combination therapy over aspirin alonecompared to the occurrence of all vascular events.

90 Ann. N.Y. Acad. Sci. 1207 (2010) 89–96 c© 2010 New York Academy of Sciences.

Melani et al. Effect of dipyridamole on spatial memory

Moreover, in ischemic stroke patients, it was demon-strated that the combination of aspirin and dipyri-damole improved neurological function better thanaspirin alone.39 From these studies, the first choicetherapy for secondary stroke prevention to emerge isthe combination of dipyridamole and aspirin.40 Therecent statement from American Heart Associationand American Stroke Association Council on Strokesays that the combination of aspirin and extended-release dipyridamole is considered safe comparedwith aspirin alone.41

Dipyridamole as neuroprotectant

Recent laboratory investigations have shown newneuroprotective properties of dipyridamole otherthan antithrombotic effects. Dipyridamole inhibitscGMP-dependent phosphodiesterase and therebypotentiates the nitric oxide system.42 It also hasantioxidant proprieties and has been described asthe most potent orally available scavenger for oxiand peroxi radicals.27,43,44 It decreases reactive oxy-gen species generation produced by activated poly-morphonuclear leukocytes (PMN)45,46 and has anti-inflammatory effects by decreasing production ofproinflammatory cytokines (TNF-�, IL-8)47 andchemokine release (MCP-1).48 By inactivating p38mitogen-activated protein kinase (MAPK) that re-duces nuclear translocation of NF-�B, dipyridamoleinhibits expression of matrix metalloprotease-9(MMP-9)48 and of COX-2 in macrophages.49 Anew view attributes anti-inflammatory actions todipyridamole by inhibition of the expression ofcritical inflammatory genes, MCP-1 and MMP-9.48

Recent evidence indicates that monocyte chemoat-tractant protein 1 (MCP-1) is essential for re-cruiting blood-borne cells to the injury site aftercerebral ischemia, whereas its deficiency does notaffect resident microglia activation and migration.50

After cerebral ischemia, microglia is rapidly acti-vated whereas hematogenous macrophage infiltra-tion is a later event. MCP-1 deficient mice,51 as wellas mice deficient of chemokine receptor 2 (CCR2)that bind MCP-1,52 show attenuated ischemic in-farct volume after transient focal ischemia. A differ-ent profile in chemokine and/or cytokines expres-sion after MCP-1 deletion could be responsible forprotection.

In recent years, several studies have suggestedthat dipyridamole can augment vessel function thusrestoring blood flow even in cerebrovascular dis-

eases.53 These effects can be accounted for by en-hancement of NO,54 VEGF production,55 and byreducing the reactive-oxygen species (ROS) sys-tem.56 Possibly secondarily to anti-inflammatory ef-fects, dipyridamole reduces neutrophil adhesion toendothelium.57

Dipyridamole administration in ananimal model

We have carried out bilateral common carotid arteryocclusion (2VO) in rats according to Sarti et al.12

Briefly, a median incision was performed in the skinof the ventral part of the neck and the subcutaneousadipose tissue was dissected, avoiding the thyroid.The omohyoid muscle was cut through with a me-dian incision. Under optic microscope, the com-mon carotid artery was visualized and exposed, sep-arating the surrounding tissue. In 30 rats, the rightcommon carotid artery was ligated with a silk su-ture firmly tied around the vessel. In 15 rats, thesame procedure was applied without the ligature ofthe right common carotid artery (sham-operated).After 1 week, the same procedure was performedin the contralateral hemisphere, occluding the leftcommon carotid artery. The model can therefore becalled that of occlusion of the two common carotidarteries separately.

Dipyridamole (Persantin, Boehringer Ingelheim,5 mg/mL) was administered by intravenous per-fusion to obtain stable and persistent concentra-tions of the drug in the blood. Dipyridamole orvehicle were injected (10 �L/h per 7 days) intothe jugular vein by a miniosmotic pump (Model2001, Alzet, Cupertino, CA, USA) attached to a sil-icone catheter (0.51 mm ID × 0.07 wall; Alzet) andprefilled with drug or vehicle in sterile conditions,followed by a 12-h preimplantation incubation at37 ◦C. The pumps were implanted subcutaneouslyin the thoracic-lumbar region immediately after2VO and removed on the 8 day (Fig. 1). Consider-ing the body weight of rats (about 300 g), each an-imal received about 4 mg/kg/day of dipyridamole.On the basis of a previous estimation of dipyri-damole plasma concentration in the rat,58 we esti-mated that administration of 4 mg/kg dipyridamolei.v. provides a 2–2.5 �M plasma concentration overthe entire week. It is worth noting that this con-centration (equivalent to 1 �g/mL) is equivalent tothe therapeutic concentration (1.6 �g/mL) reachedafter extended release dipyridamole is administered

Ann. N.Y. Acad. Sci. 1207 (2010) 89–96 c© 2010 New York Academy of Sciences. 91


Figure 1. Experimental protocol utilized in the separate 2VO model at an interval of 1 week. Dipyridamole was administeredintravenously by miniosmotic pump. Cognitive behavior and sensory motor tests were evaluated 1–3 months after 2VO.

to ischemic stroke patients.59 A lethal dipyridamoledose in the rat is estimated to be 200 mg/kg i.v.58

The dose of dipyridamole administered in this studyis similar to that (0.5–3 mg/kg i.v.) used by Joneset al.60 In this range, dipyridamole dose dependentlyincreases coronary blood flow velocity during timeof infusion. In the study of Hung et al.,61 dipyri-damole (1.4 mg/die/kg i.v. per 7 days) decreases ratperitoneal fibrosis.

Histological analysis after 2VO

Three months after occlusion, we did not find anysignificant ischemic brain damage by Fluoro-JadeB (FJB) staining. The lack of significant neuronaldeath 3 months after 2VO was confirmed in our ex-periments by positive cresyl violet staining. Theseresults are in agreement with the lack of neuronaldamage found by FJB staining 2–6 months afterpermanent 2VO.11,62 In the 2VO model, however,some damage (modest and not in all animals) tohippocampal neurons was found 1–2 weeks afterpermanent and transient 2VO, respectively, by cre-syl violet staining63,64 or Tunel assay.65,66 Three daysafter transient (20 min) 2VO, laminin degrada-tion was reported in both CA1 and CA2 pyrami-dal neuronal layers.67 A modest but significant cellloss was reported in the hippocampus from 2 to

6 months after permanent 2VO by hematoxylin–eosin staining.11,62

The lack of significant neuronal damage foundin our experiments is in agreement with the lack ofdamage, evaluated by hematoxylin–eosin, 3 monthsafter occlusion reported by Sarti et al.12 The occlu-sion model of the two common carotid arteries sep-arately at an interval of 1 week, as in the work by Sartiet al.12 as well as in our work, should allow hypop-erfusion to develop more gradually followed by lessneuronal death. In agreement with this, rat modelsof chronic ischemia have shown that neurons arecapable of existing in a suspended metabolic statewhen a diminished energy substrate is available,68

such as a prolonged hypometabolism state that canexist in viable cells.

In our immunohistochemical experiments,myelin organization and vascular density, evaluatedby an antibody against myelin-associated glycopro-tein (MAG) and an antibody against laminin, whichis a complex extracellular glycoprotein-abundantcomponent of endothelium basement membranes,respectively, did not show differences between 2VO-and sham-operated rats. No astrogliosis was de-tected by an antibody against glial fibrillary acidicprotein (GFAP), while a certain degree of microglialreactivity, evaluated by an antibody against ionizedcalcium binding adaptor molecule 1 (IBA-1), was



Figure 2. Sensorimotor evaluation over time of sham- (•),vehicle- (◦), and dipyridamole- (�) treated rats. The mean ±SEM of values from 14 to 90 days after 2VO is presented forall three rat groups. Two-way ANOVA followed by Bonferronimultiple comparison test: §P < 0.05 vs. sham; #P < 0.01 vs.sham.

present or persisted for 3 months after 2VO in thehippocampus. At this time, microglia reactivity wasnot modified in dipyridamole-treated rats.

Dipyridamole and spatial working memory

In 2VO rats, a significant neurological deficit eval-uated by a battery of six sensory-motor tests69 ispresent 2–3 months after 2VO. Small animals areknown to recover from neurological deficit moreeasily than humans, even within hours or days afterischemia. After transient ischemia induced by me-dial cerebral artery occlusion, animals tend to re-cover their sensory-motor functions in 1 week.69,70

On the contrary, in the present hypoperfusionmodel, sensory-motor functions tended to decreaseover time, arriving at significant impairment 2 and3 months after 2VO. This demonstrates a slow de-terioration of sensory-motor functions followingchronic hypoperfusion. Dipyridamole did not sig-nificantly ameliorate neurological deficit (Fig. 2).

Two and three months after 2VO, rats also hada significant deficit of spatial working memory, asshown by a decreased alternance in the Y maze test,while the nonspatial memory measured by the ob-ject recognition test was not significantly affected.

Disruption of complex behaviors may reflectalterations at the subcellular, synaptic, or elec-trophysiological levels, or even of widespreadmorphological changes that cannot be quantified

histologically,71 as found in our study. A reductionin cerebral metabolism is sufficient to induce animpairment of working memory.72

Dipyridamole, administered for a week into thejugular vein, significantly restores spatial memory,that is, the alternance in the Y maze test (Fig. 3), tovalues similar to those found in sham-operated rats3 months after 2VO. Interestingly, Weinstock andShoham73 demonstrated that it is possible to rescuehippocampal neurons and prevent spatial memorydeficits if the vessels are deoccluded 1 or 2 weekslater.

The general protective effect of dipyridamole wasalso illustrated by significant restoration of rat bodyweight 2 and 3 months after 2VO (Fig. 4).

Therefore, dipyridamole is able to improve cog-nitive performance without ameliorating sensori-motor functions. Data from neuroprotective drugstudies in animal models of ischemia suggest a poorcorrelation between pathologic and functional im-provement.74 For instance, despite the lack of a clearpathological evidence of infarct size improvement,behavioral assessment might reveal the effectivenessof a neuroprotective drug.75,76 Lack of correlationbetween different outcome measures indicates thatboth behavioral, neurological, and histological endpoints are necessary for effectively and compre-hensively examining the putative protective effectof a drug in models of stroke but also in chronic

Figure 3. Spatial working memory evaluation over time ofsham- (•), vehicle- (◦), and dipyridamole- (�) treated rats.The mean ± SEM of values from 14 to 90 days after 2VO ispresented for all three rat groups. Two-way ANOVA followed byBonferroni multiple comparison test: #P < 0.05 vs. sham; §P <

0.05 vs. dipyridamole.



Figure 4. Body weight over time of sham- (•), vehicle- (◦),and dipyridamole- (�) treated rats. The mean ± SEM of valuesfrom 14 to 90 days after 2VO is presented for all three rat groups.Two-way ANOVA followed by Bonferroni multiple comparisontest: #P < 0.05 vs. sham.

cerebral hypotension. Therefore, combining appro-priate behavioral tests with histological measure-ments becomes more and more critical in neu-rorestorative drug studies.77,78

Conclusions

Dipyridamole, chronically administered for 1 week,improves the spatial working memory 3 months af-ter chronic hypoperfusion induced by 2VO. At thistime, dipyridamole probably improved neurobe-havior secondarily to anti-inflammatory effects. Onthese bases, in further studies it will be interestingto investigate if dipyridamole’s protective effects oncognition, observed in our chronic cerebral ischemicmodel, are correlated with reduced inflammationparameters that occur between 1 and 2 monthsafter 2VO.

Acknowledgments

This work was financed by Boehringer Ingelheimand Ente Cassa di Risparmio di Firenze.

Conflicts of interest

This research was partly founded by BoehringerIngelheim.

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Effect of intravenous administration of dipyridamole in a rat model of chronic cerebral ischemia