ARTICLE IN PRESS

Dipyridamole plus Triflusal versus Triflusal Alone in Infarct

Reduction after Middle Cerebral Artery Occlusion

Zareen Amtul, PhD,* Wasimul Haque, PhD,† and David F. Cechetto, PhD*

Background and Purpose: The objective of this work is to study the dose-
dependent effect of combination therapy with dipyridamole and triflusal over that
of triflusal alone on infarct size after middle cerebral artery occlusion (MCAO)
ischemia. Materials and Methods: Male Wistar rats were subjected to a perma-
nent MCAO in the right hemisphere. Rats received triflusal alone and with
dipyridamole via oral route. Three days after surgery, infarct volumes were
measured. Results: The lower dose regime of triflusal (10 mg/kg) and
dipyridamole (200 mg/kg) caused the greatest decrease in infarct size compared
with higher dose regime of triflusal (30 mg/kg) and dipyridamole (200 mg/kg)
(P < .01), triflusal (30 mg/kg) alone (P < .07), and vehicle-treated controls. Conclu-
sions: The lower dose combination of dipyridamole and triflusal appears to be
more effective than triflusal alone after MCAO-induced cerebral ischemia.
Therefore, there is a strong rationale to continue to examine the protective effects
of triflusal and dipyridamole after cerebral ischemia. Key Words:
Ischemia—MCAO—dipyridamole—triflusal—infarct.
© 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Introduction States.1 There are 2 major types of stroke pathogenesis:

Epidemiologically, neural stroke remains the most fre-
quent cause of disability and death in industrialized and
developing countries, after cancer and heart disease. Stroke
is also the reason why the majority of patients suffer and
are hospitalized from acute brain syndromes in the United
From the *Anatomy and Cell Biology Department, The Univer-
sity of Western Ontario, London, Ontario, Canada; and †Kardiatech
Inc., Edmonton, Alberta, Canada.
Received October 23, 2017; revision received November 16, 2017;
accepted December 11, 2017.
Financial disclosure: Wasimul Haque is the chief scientific officer
and part owner of Kardiatech Inc. However, he had no role in the
conception, study design, conduct of the experiment, data collec-
tion, analysis, interpretation, or preparation of the manuscript.
Kardiatech Inc. holds the provisional patent (WO2011017810 A1) for
triflusal and dipyridamole combination therapy. The rest of the authors
declare that they have no conflict of interest.
Address correspondence to Zareen Amtul, PhD, Anatomy and Cell
Biology Department, The University of Western Ontario, London,
Ontario, Canada N6A 5C1. E-mail: zareen.amtul@gmail.com.
1052-3057/$ - see front matter
© 2017 National Stroke Association. Published by Elsevier Inc. All
rights reserved.
https://doi.org/10.1016/j.jstrokecerebrovasdis.2017.12.013
transitory or permanent occlusion of cerebral blood vessels
or ischemic (more than 80% of strokes are ischemic) and
hemorrhagic.2 Both types result in subsequent tissue loss
with sustainable behavioral deficits.2-4 The middle cere-
bral artery (MCA) is the most universally affected blood
vessel in acute ischemic disorders5 and the most widely
coagulated or occluded blood vessel.6 By inserting an in-
traluminal suture7 in the experimental rodent focal ischemia
model, MCA occlusion (MCAO) produces a well-
defined region of injury that includes the neocortex and
the lateral striatum, resulting in underlying functional
deficits.8
These facts make it critical to develop primary and
secondary strategies to prevent or treat stroke. There
are 2 major therapeutic approaches aimed at preventing
or intervening after ischemia: reperfusion therapy and
antiplatelet therapy. Reperfusion therapy restores blood
flow through blocked arteries after stroke either by
clot-busting (thrombolytic) drugs, by opening arteries
with stents, or by grafting arteries around blockages.
Tissue plasminogen activator efficiently improves clini-
cal outcomes in reperfusion therapy after cerebral
ischemia. However, in spite of its high efficacy, tissue
plasminogen activator needs to be used within hours

Journal of Stroke and Cerebrovascular Diseases, Vol. ■■, No. ■■ (■■), 2017: pp ■■–■■ 1
 ARTICLE IN PRESS
2 Z. AMTUL ET AL.
9
after the ischemia onset with a related risk of hemor-
rhagic transformation.10 Antiplatelet therapy decreases
platelet aggregation and inhibits thrombus formation.
Antiplatelet therapy, in addition to lifestyle changes,
is the basis of arterial thrombotic stroke prevention
in the secondary setting. Antiplatelet therapy also
improves cardiovascular risk. Even though acetylsali-
cylic acid has proved its effectiveness in preventing
atherothrombotic episodes, there are key limitations of
this drug including the high risk of hemorrhage,
limited efficacy, significant variability in interindividual
response, and extended length of action that cannot
be reversed if emergency surgery or hemostasis is
needed.11
Unfortunately, newer antiplatelet therapies do not
offer considerably improved protection over and above
acetylsalicylic acid to treat atherothrombotic stroke.
Thus, there is a need to identify and formulate new
antiplatelet therapies that are appropriate for use in a
larger population of stroke victims, either alone or in
combination with reperfusion treatments.12 Triflusal (2-
acetyloxy-4-trifluoromethyl benzoic acid), a 4-fluoromethyl
member of the salicylate family, directly inhibits
cyclooxygenase-2 and irreversibly and selectively inhib-
its cyclooxygenase-1 and arachidonic acid metabolism
in platelets. Triflusal is an indirect inhibitor of nuclear
factor κB13 and inhibits platelet aggregation, thus
represents a promising substitute for acetylsalicylic
acid, while presenting a more favorable safety profile.
Triflusal at a dose of 30 mg/kg has been demonstrated
to be effective in reducing infarct size in the rat
MCAO stroke model.14 Dipyridamole (2-{[9-(bis(2-
hydroxyethyl)amino)-2,7-bis(l-piperidyl)-3,5,8,10-
tetrazabicyclo [4.4.0]deca-2,4,7,9,11-pentaen-4-yl]-(2-
hydroxyethyl)amino}ethanol) was introduced into clinical
practice in the early 1960s as a cardioprotective coro-
nary vasodilator.15 Dipyridamole has both antiplatelet
and vasodilatory properties with a mechanism of action
that is probably related to the inhibition of platelet
phosphodiesterase, stimulation of prostacyclin release,
or inhibition of adenosine uptake.16 Dipyridamole has
been shown to be protective against cognitive impair-
ment after global ischemia.17
The lone use of triflusal or a combined regimen of
dipyridamole and triflusal treatment is more effective
in the secondary prevention of atherothrombotic stroke
than acetylsalicylic acid and dipyridamole combination.18
We therefore hypothesize that the combination of sub-
optimal doses of dipyridamole and triflusal will provide
neuroprotection by decreasing infarct size after MCAO
compared with triflusal alone. At present, we aim to
investigate whether acute poststroke use of dipyridam-
ole and triflusal, which is very similar to a clinical
experimental paradigm for secondary stroke preven-
tion, offers any improvement to ischemic cerebral
injury.
Materials and Methods
Animal, Treatment, and Tissue Preparation
All animal protocols were carried out according to the
guidelines of the Animal Use and Care Committee of
Western University (approval ID: 2008-113). Male Wistar
rats (265-36 g) were anesthetized using a single intra-
peritoneal dose of sodium pentobarbital (60 mg/kg). The
animals were positioned in a stereotaxic apparatus (David
Kopf Instruments, Tujunga, CA) with the incisor bar below
the interaural line, set at 3.3 mm. Body temperatures were
maintained at 37°C. Before and after surgery, rats were
housed in single cages (12/12-hour light/dark cycle) and
were fed ad libitum. To model ischemic stroke, the right
MCA was exposed and permanently occluded at 2 points,
1 above and 1 below the inferior cerebral vein, using a
Helica thermal coagulator (a combination of helium gas
and a low alternating current of 50 W). The removal of
a portion of bone and the exposure and ligation of the
MCA were done with the aid of a surgical operating mi-
croscope (stereomicroscope; Leica, MZ6, Wetzlar, Germany).
After suturing the wound, all rats received a subcuta-
neous injection of 30 µg/kg buprenorphine and an
intramuscular injection of 20 µL (50 mg/mL stock) of
enrofloxacin antibiotic (Baytril; Bayer Inc., Toronto, ON,
Canada). The rat model of MCAO is well established in
our laboratory. There were 4 groups of animals (n = 8 for
each group). Immediately after surgery and for 2 addi-
tional days, rats received one of the following treatment
suspensions via oral gavage: 200 mg/kg dipyridamole plus
10 mg/kg triflusal (D + T10 group), 200 mg/kg dipyri-
damole plus 30 mg/kg triflusal (D + T30 group), or 30 mg/
kg triflusal (T30 group). The control rats (control group)
went through the identical steps but only received the
vehicle (analytical-grade ethanol) via oral gavage. Three
days after surgery, rats were euthanized with an over-
dose of sodium pentobarbital (160 mg/kg, intraperitoneal)
and transaortically perfused, first with heparinized
phosphate-buffered saline followed by a slow
perfusion for about 1 hour with 2% solution of 2,3,5-
triphenyltetrazolium chloride (Sigma-Aldrich, St. Louis,
MO)18 and then followed by 4% formaldehyde (pH 7.4).
The brains were immediately removed and sliced 1.0 mm
apart, with the help of a slicer matrix. At the time of
surgery, gross cerebral hemorrhage was noticed in 3 rats;
these rats were replaced at a later date to maintain the
same number in each group. No mortality occurred.
Analyses
Anterior images of the stained brain slices were cap-
tured using a Nikon Digital Camera (COOLPIX P80 Tokyo,
Japan) with 10.1 megapixel and 18× optical zoom, powered
by a large 1/1.8-inch format, high-resolution liquid-
crystal display (LCD). Serial brain sections were examined,
and the infarcted tissue areas were measured and
 ARTICLE IN PRESS
DIPYRIDAMOLE AND TRIFLUSAL
quantified using an image analysis system (SigmaScan
Pro 5.0; SPSS Inc., Chicago, IL). In addition, the hemi-
spheric areas of each brain slice were measured to account
for any swelling of brain that might have resulted due
to MCAO. The infarct area was multiplied by the ratio
of the contralateral area to the ipsilateral area to correct
for brain edema.19 The volumes of the infarcts were cal-
culated in mm3 by integrating the infarct sizes for each
of the brain slices. The average of the counts done by 2
blinded scientists was presented as mean ± standard error
of the mean. All measurements were analyzed using one-
way analysis of variance followed by post hoc Dunnett
tests. The significance level was P ≤ .05.
Results
Effects of Dipyridamole and/or Triflusal on Infarct Size
In vehicle-treated control rats, the clear border of the
infarct was evident 3 days after MCAO. Briefly, the uni-
lateral coagulation of MCA in the right or ipsilateral
3
hemisphere produced infarcts that were restricted to the
right cortex only. The contralateral or left hemisphere did
not show any injury. The administration of dipyridam-
ole plus triflusal (P < .01) and triflusal alone (P < .01) (Fig 1)
triggered a significant reduction in the total infarct volume
in MCA-occluded rats compared with the vehicle-
treated control group (Fig 2). However, the combination
of dipyridamole plus lower dose triflusal was more ef-
fective than triflusal alone.
The infarct volume in rats that received vehicle treat-
ment 3 days after MCAO was 106.22 ± 10 mm3; in the
animals treated with D + T10, it was 62.58 ± 4.0 mm3; in
the animals treated with D + T30, it was 81.13 ± 5.3 mm3;
and in the animals treated with T30, it was 73.59 ± 7.0 mm3
(Table 1). The reduction in infarct volume was up to 37.03%
in D + T10, 23.62% in D + T30, and 30.72% in T30 alone
(in agreement with our previous findings after MCAO14)
compared with vehicle-treated controls. Intriguingly, the
D + T10–treated animals displayed significantly better effects
on infarct size than the D + T30 and T30 alone rats.

Figure 1. Dipyridamole and triflusal: Schematic rep-

resentation of triflusal and dipyridamole structures. The
chemical synthesis of triflusal, having a trifluoromethyl
group in position 4, is different but structurally related
to acetylsalicylic acid. The chemical formula of dipyri-
damole is 2-{[9-(bis(2-hydroxyethyl)amino)-2,7-bis(l-
piperidyl)-3,5,8,10-tetrazabicyclo [4.4.0]deca-2,4,7,9,
11-pentaen-4-yl]-(2-hydroxyethyl)amino}ethanol.

Figure 2. Effects of dipyridamole and/or triflusal on

infarct size: Representative coronal sections stained with
2,3,5-triphenyltetrazolium chloride, 3 days after middle
cerebral artery occlusion show cortical lesions at bregma
level 0.48 mm in vehicle-treated (control), 200 mg/kg
dipyridamole and 10 mg/kg triflusal-treated (D + T10),
200 mg/kg dipyridamole and 30 mg/kg triflusal-
treated (D + T30), and 30 mg/kg triflusal-treated (T30)
rats. The plot shows the measured infarct volume in
control, D + T10, D + T30, and T30 rats, **P < .01.
 ARTICLE IN PRESS
4 Z. AMTUL ET AL.
Table 1. Effects of vehicle (control), triflusal (T), and dipyridamole (D) on infarct volumes 3 days after unilateral middle cerebral
artery occlusion

Treatment Infarct volume (mm3) Infarct size reduction (%)

Control (vehicle) 106.22 ± 10 0

D + T10 (200 mg/kg dipyridamole + 10 mg/kg triflusal) 62.58 ± 4.0 37.03 ± 7.19
D + T30 (200 mg/kg dipyridamole + 30 mg/kg triflusal) 81.13 ± 5.3 23.62 ± 4.95
T30 (30 mg/kg triflusal) 73.59 ± 7.0 30.72 ± 6.6

Discussion and dipyridamole presently demonstrated, further in-

vestigation is needed to establish if dipyridamole and
In the present study, we demonstrated that dipyri-
triflusal after ischemia combined with thrombolytic treat-
damole plus triflusal or triflusal alone significantly reduced
ments represents the best acute treatment for patients with
the infarct size compared with vehicle-treated animals,
cerebral ischemia. The translation of dipyridamole and
3 days after MCAO, when administered concurrent with
triflusal to clinical trials to examine its efficacy, when ad-
cerebral ischemia via oral gavage. Our results also suggest
ministered immediately after the onset of stroke symptoms,
that the dipyridamole plus lower triflusal dose (10 mg/
appears feasible, given the fact that dipyridamole is pres-
kg) regimen is more effective than the dipyridamole plus
ently widely administered clinically for the prevention
higher triflusal dose (30 mg/kg) or triflusal alone treat-
of secondary stroke.
ments. Moreover, dipyridamole plus triflusal or triflusal
Consequently, the present study demonstrates the use-
alone produced what is likely the maximum infarct re-
fulness of dipyridamole plus triflusal to provide an infarct
duction in this type of stroke model. The combination
reduction effect. We also propose that other techniques
of dipyridamole with triflusal shows added synergism
and markers of injury assessment, such as histology,22,23
when the lower dose of triflusal (10 mg/kg) was used.
hematoxylin and eosin, imaging,24 hemodynamics, cere-
These preliminary results certainly suggest that the com-
bral perfusion,25 and blood–brain barrier disruption,26,27
bination of dipyridamole and triflusal is as effective as
may be beneficial for showing different aspects of
reported for dipyridamole and acetylsalicylic acid.20 As
neuroprotection. To provide more correlation with his-
triflusal has a lower hemorrhagic effect than acetylsali-
topathology and to determine the effects of drugs on the
cylic acid and is a more direct inhibitor of nuclear factor
neuroinflammatory pathways, we suggest using longer
κB, 13 it is a better drug candidate to combine with
time points. At present, it is, however, not yet certain if
dipyridamole.
the 3-day infarct reduction timeline after triflusal and di-
Triflusal alone resulted in a 30% decrease in infarct size
pyridamole treatment does represent the maximum infarct
(the terms infarct size and infarct volume are used in-
reduction. An understanding of the critical window for
terchangeably). This is an excellent decrease in infarct size
therapeutic manipulation can be enhanced with such an
representing considerable stroke prevention. However, it
investigation.
is hard to expect more than 30% reduction in infarct size
after only 3 days of oral treatment; it may be the most
Acknowledgment: This research was partly funded by a
that can be observed based on our experience and other
grant from the Kardiatech Inc.
investigations.14 The present results, however, indicate that
a combination of dipyridamole with a lower concentra-
tion of triflusal is an even more effective treatment strategy References
based on their mechanisms of neuroprotection, presum-
1. Zhang Y, Chapman A-M, Plested M, et al. The incidence,
ably, due to the weak clinically relevant pharmacodynamics prevalence, and mortality of stroke in France, Germany,
or pharmacokinetics drug interactions and strong syn- Italy, Spain, the UK, and the US: a literature review. Stroke
ergistic interactions when lower concentration of triflusal Res Treat 2012;2012:436125.
was used. In the present study, we also established that 2. Mitsios N, Gaffney J, Kumar P, et al. Pathophysiology
oral gavage is an optimal method of acute dipyridam- of acute ischaemic stroke: an analysis of common
signalling mechanisms and identification of new molecular
ole administration and avoids lethal side effects that are targets. Pathobiology 2006;73:159-175.
seen via intravenous administration of dipyridamole.21 3. Lo EH, Dalkara T, Moskowitz MA. Neurological diseases:
Moreover, it is appealing to speculate that the current mechanisms, challenges and opportunities in stroke. Nat
standard stroke preventive measures could be im- Rev Neurosci 2003;4:399-415.
proved if dipyridamole were combined with existing 4. Mergenthaler P, Dirnagl U, Meisel A. Pathophysiology
of stroke: lessons from animal models. Metab Brain Dis
thrombolytic therapies, provided the previous experi- 2004;19:151-167.
mental setup is translated to the clinical use. However, 5. Carmichael ST. Rodent models of focal stroke: size,
despite the protective effect of the combination of triflusal mechanism, and purpose. NeuroRx 2005;2:396-409.
 ARTICLE IN PRESS
DIPYRIDAMOLE AND TRIFLUSAL
6. Howells DW, Porritt MJ, Rewell SSJ, et al. Different
strokes for different folks: the rich diversity of animal
models of focal cerebral ischemia. J Cereb Blood Flow
Metab 2010;30:1412-1431.
7. Kawamura S, Yasui N, Shirasawa M, et al. Rat middle
cerebral artery occlusion using an intraluminal thread
technique. Acta Neurochir (Wien) 1991;109:126-132.
8. Ginsberg MD, Busto R. Rodent models of cerebral
ischemia. Stroke 1989;20:1627-1642.
9. Hacke W, Kaste M, Bluhmki E, et al. Thrombolysis with
alteplase 3 to 4.5 hours after acute ischemic stroke. N
Engl J Med 2008;359:1317-1329.
10. The NINDS t-PA Stroke Study Group. Intracerebral
hemorrhage after intravenous t-PA therapy for ischemic
stroke. Stroke 1997;28:2109-2118.
11. Eikelboom JW, Hirsh J, Spencer FA, et al. Antiplatelet
drugs: antithrombotic therapy and prevention of
thrombosis, 9th ed: American College of Chest Physicians
Evidence-Based Clinical Practice Guidelines. Chest
2012;141:e89S-e119S.
12. Amtul Z, Rahman AU. Neutraceuticals neuroprotect
naturally: Alzheimer’s disease, Parkinson’s disease, stroke
and major depressive disorder. In: Rahman A, ed. Studies
in natural product chemistry. Elsevier Science Publishers,
2017:373-397.
13. Bayón Y, Alonso A, Crespo MS. 4-Trifluoromethyl
derivatives of salicylate, triflusal and its main metabolite
2-hydroxy-4-trifluoromethylbenzoic acid, are potent
inhibitors of nuclear factor κB activation. Br J Pharmacol
1999;126:1359-1366.
14. Figueredo VM, Diamond I, Zhou HZ, et al. Chronic
dipyridamole therapy produces sustained protection
against cardiac ischemia-reperfusion injury. Am J Physiol
1999;277:H2091-H2097.
15. Sudlow C. What is the role of dipyridamole in long-term
secondary prevention after an ischemic stroke or transient
ischemic attack? Can Med Assoc J 2005;173:1024-1026.
16. Melani A, Cipriani S, Corti F, et al. Effect of intravenous
administration of dipyridamole in a rat model of
chronic cerebral ischemia. Ann N Y Acad Sci 2010;
1207:89-96.
5
17. Guiteras P, Altimiras J, Arís A, et al. Prevention of
aortocoronary vein-graft attrition with low-dose aspirin
and triflusal, both associated with dipyridamole:
a randomized, double-blind, placebo-controlled trial. Eur
Heart J 1989;10:159-167.
18. Bederson JB, Pitts LH, Germano SM, et al. Evaluation
of 2,3,5-triphenyltetrazolium chloride as a stain for
detection and quantification of experimental cerebral
infarction in rats. Stroke 1986;17:1304-1308.
19. Burguete MC, Torregrosa G, Pérez-Asensio FJ, et al.
Dietary phytoestrogens improve stroke outcome after
transient focal cerebral ischemia in rats. Eur J Neurosci
2006;23:703-710.
20. Halkes PHA, Gray LJ, Bath PMW, et al. Dipyridamole
plus aspirin versus aspirin alone in secondary prevention
after TIA or stroke: a meta-analysis by risk. J Neurol
Neurosurg Psychiatry 2008;79:1218-1223.
21. García-Bonilla L, Sosti V, Campos M, et al. Effects of acute
post-treatment with dipyridamole in a rat model of focal
cerebral ischemia. Brain Res 2011;1373:211-220.
22. Amtul Z, Hepburn JD. Protein markers of cerebrovascular
disruption of neurovascular unit: immunohistochemical
and imaging approaches. Rev Neurosci 2014;25:481-
507.
23. Amtul Z, Whitehead SN, Keeley RJ, et al. Comorbid rat
model of ischemia and β-amyloid toxicity: striatal and
cortical degeneration. Brain Pathol 2015;25:24-32.
24. Amtul Z, Nikolova S, Gao L, et al. Comorbid Aβ toxicity
and stroke: hippocampal atrophy, pathology, and cognitive
deficit. Neurobiol Aging 2014;35:1605-1614.
25. Yang J, D’Esterre CD, Amtul Z, et al. Hemodynamic
effects of combined focal cerebral ischemia and amyloid
protein toxicity in a rat model: a functional CT study.
PLoS ONE 2014;9:e100575.
26. Amtul Z, Yang J, Nikolova S, et al. The dynamics of BBB
breakdown in a rat model of beta-amyloid toxicity and
ischemia. Molecular Neurobiology (accepted). 2017/2018.
27. Amtul Z, Frías C, Randhawa J, et al. The spatial cerebral
damage induced by the higher dose of endothelin-1 and
beta-amyloid toxicity is driven by the anatomical
connectivity of CNS. Submitted. 2018.