Ebselen, a Seleno-Organic Antioxidant, Is Neuroprotective

After Embolic Strokes in Rabbits

Synergism With Low-Dose Tissue Plasminogen Activator
Paul A. Lapchak, PhD; Justin A. Zivin, MD, PhD

Background and Purpose—It has been proposed that antioxidants and spin-trap agents may be neuroprotective after acute
ischemia stroke. Although the antioxidant ebselen is currently in clinical trials, little is known about the effectiveness
of ebselen, which has glutathione peroxidase–like and anti-inflammatory properties in embolic stroke models.
Therefore, we determined the effects of ebselen when administered alone or with the thrombolytic tissue plasminogen
activator (tPA), the only Food and Drug Administration–approved pharmacological agent for the treatment of stroke.
Methods—Male New Zealand White rabbits were embolized by injection of a suspension of small blood clots into the
middle cerebral artery via a catheter. Five minutes after embolization, ebselen (10 to 50 mg/kg) was infused
intravenously. Control rabbits received infusions of the vehicle required to solubilize ebselen. In additional rabbits,
ebselen (20 mg/kg) was administered 60 minutes after embolization, either alone or in combination with tPA (0.9 or 3.3
mg/kg tPA). Behavioral analysis was conducted 24 hours after embolization, allowing determination of the effective
stroke dose (P50) or clot amount (mg) that produces neurological deficits in 50% of the rabbits.
Results—A drug is considered neuroprotective if it significantly increases the P50 compared with the vehicle-treated control
group. The P50 of controls 24 hours after embolization was 1.35⫾0.30 mg. Rabbits treated 5 minutes after embolization
with 10, 20, or 50 mg/kg ebselen had P50 values of 2.12⫾0.56, 2.82⫾0.75 (P⬍0.05), and 0.49⫾0.54 mg, respectively.
A significant neuroprotective effect was observed with the 20-mg/kg dose, but not if there was a 60-minute delay before
administration (P50⫽1.69⫾0.32 mg). When tPA (3.3 mg/kg) was infused 60 minutes after embolization and ebselen (20
mg/kg) was injected at either 5 (P50⫽2.98⫾0.18 mg) or 60 (P50⫽3.60⫾0.79 mg) minutes, there was no additional
neuroprotective effect compared with tPA alone (P50⫽3.38⫾0.55 mg). However, if ebselen (20 mg/kg) was
administered concomitantly with low-dose tPA (0.9 mg/kg) 60 minutes after embolization, the P50 was 3.52⫾0.73 mg
(P⬍0.05), indicating a synergistic effect of the drug combination because neither alone was effective (P50⫽1.69⫾0.32
and 1.54⫾0.36 mg, respectively).
Conclusions—This study indicates that ebselen may be neuroprotective when administered shortly after an embolic stroke,
but the time- and dose-response analyses suggest that it has a narrow therapeutic window. Nevertheless, ebselen may
be beneficial if administered concomitantly with a thrombolytic because it significantly enhanced the neuroprotective
activity of low-dose tPA. (Stroke. 2003;34:2013-2018.)
Key Words: ischemia 䡲 neuroprotection 䡲 reperfusion 䡲 thrombolytic therapy 䡲 tissue plasminogen activator 䡲 rabbits

E bselen [2-phenyl-1,2-benziselenazol-3(2H)-one] is a
selenium-based antioxidant compound with numerous
pharmacological activities,1 including neuroprotection medi-
ated by REDOX reactions,2 reduced inflammatory mecha-
nisms,1 scavenging of reactive nitrogen species including
peroxynitrite,3–5 and inhibition of apoptotic mechanisms.6
More important, recent studies using models of neurodegen-
erative diseases have shown that ebselen can modify
N-methyl-D-aspartate (NMDA) receptor function, reducing
the devastating effects of NMDA receptor activation.2 Addi-
tionally, ebselen neutralizes free radicals produced by NMDA
receptor stimulation7 and reduces lipoperoxidation mediated
by glutamate-induced excitotoxicity.8 This represents an im-
portant property of ebselen because free radicals, particularly
reactive oxygen species, can accumulate to such a degree as
to cause a biochemical chain of events that ultimately lead to
cell death.9
Preliminary clinical trials have shown that ebselen may be
useful in the treatment of acute ischemic stroke. The first
report of a placebo-controlled, double-blind clinical trial
showed that there was a significantly better outcome in
ebselen-treated patients at 1 month but not 3 months after

Received December 11, 2002; final revision received March 11, 2003; accepted April 9, 2003.
From the University of California San Diego, Department of Neuroscience, La Jolla; and VASDHS and Veterans Medical Research Foundation, San
Diego, Calif.
Correspondence to Dr Paul A. Lapchak, University of California San Diego, Department of Neuroscience, MTF 316, 9500 Gilman Dr, La Jolla, CA
92093– 0624. E-mail plapchak@ucsd.edu
© 2003 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org DOI: 10.1161/01.STR.0000081223.74129.04

2013
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2014 Stroke August 2003
treatment.10 A second report concluded that ebselen slightly
reduced cerebral infarct volume, but the magnitude of the
neuroprotection was not statistically significant.11 In fact, a
good outcome was seen in only ⬇15% of patients. Therefore,
although the results are promising, ebselen alone may not be
an optimal treatment to achieve significant clinical improve-
ment in patients with severe acute ischemic stroke. This is not
surprising given that, with the exception of thrombolysis,
monotherapy for stroke has proven unsuccessful (see
reviews12–14).
The main objective of the present study was to assess the
neuroprotective profile of ebselen in a rabbit model of
embolic stroke that reproduces many facets of human
stroke.15–17 The rabbit small clot embolism model (RSCEM)
uses administration of sized blood microclots to induce
strokes and behavioral deficits that can be quantified, result-
ing in random infarcts throughout the brain.15,16 Moreover,
the RSCEM is useful in testing the effects of drugs, whether
alone or in combination with the only Food and Drug
Administration–approved therapy for stroke, the thrombolytic
tissue plasminogen activator (tPA). This makes the RSCEM
ideally suited to assess whether the combination of 2 drugs
(ebselen and tPA) with radically different mechanisms of
action interact to enhance behavioral outcome.
Methods
The procedures used in this study were approved by the Department of
Veterans Affairs and the subcommittee on animal studies at the VA San
Diego Healthcare System (VASDHS). Throughout the study, the health
status of the rabbits was closely monitored by the veterinarian and staff
on duty at the VASDHS.
Male New Zealand White rabbits were anesthetized with halo-
thane (5% induction, 2% maintenance by face mask); the bifurcation
of the right carotid artery was exposed; and the external carotid
artery was ligated just distal to the bifurcation, where a catheter was
inserted anteriorly into the common carotid and secured with
ligatures. The incision was closed around the catheter with the distal
ends left accessible outside the neck; the catheter was filled with
heparinized saline and plugged with an injection cap. Rabbits were
allowed to recover from anesthesia for a minimum of 3 hours until
they awoke and behaved normally.
For the RSCEM, microclots were prepared from blood drawn
from a donor rabbit and allowed to clot at 37°C as described in detail
previously.15,16 To assess the hemorrhage incidence with low- (0.9
mg/kg) and high- (3.3 mg/kg) dose tPA, we used the rabbit large clot
embolism model (RLCEM). For RLCEM experiments, blood clots
were prepared and injected as described previously.15,18
Drug Administration
Drugs were given intravenously starting 5 or 60 minutes after
embolization. Ebselen (10, 20, or 50 mg/kg), a gift from DAIICHI
Pharmaceuticals or purchased from Alexis Biochemical, was sus-
pended in 50% ␤-hydroxypropyl cyclodextrin (Cerestar Inc) for
injection.
tPA (3.3 or 0.9 mg/kg) was given 60 minutes after embolization,
with 20% as a bolus injection over 1 minute, followed by the
remainder infused over 30 minutes.18 Genentech, Inc supplied tPA
lyophilized in 50-mg configurations containing 50 mg tPA (29
million IU), 1.7 mg L-arginine, 0.5 g phosphoric acid, and ⬍4 mg
Polysorbate 80, the same formulation used clinically, that was then
reconstituted with sterile water (1 mg/mL).
For combination studies, ebselen (20 mg/kg) was infused begin-
ning 5 or 60 minutes after embolization, and tPA was given (3.3 or
0.9 mg/kg) beginning 60 minutes after embolization.
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Rabbits were observed continuously for a minimum of 2 hours
after embolization and treatment; neurological function was scored
than and again at 24 hours after embolization. For the 24-hour score,
an observer who was naive to the treatments did the end-point
analysis.
RSCEM Analyses
For the RSCEM, a quantal dose-response data analysis technique
was used as described previously15,16 and reviewed in detail by Zivin
and Waud.17 A wide range of clot doses were used, resulting in
normal and abnormal animals, with small numbers of microclots
causing no grossly apparent neurological dysfunction and large
numbers of microclots invariably causing encephalopathy or death.
Using a simple dichotomous rating system with a reproducible
composite result and low interrater variability (⬍5%), a naive
observer rated each animal as either behaviorally normal or abnor-
mal. Abnormal rabbits include those with ⱖ1 of the following
symptoms: ataxia, leaning, circling, lethargy, nystagmus, loss of
balance, loss of sensation, and occasionally paraplegia. Using the
system, we are unable to detect small changes in improvement over
the duration of the study, which may be considered a type 2 error
because of reduced power to detect small changes. However, the
model and analysis are well suited to detect robust changes after
pharmacological intervention. Moreover, because of the use of a
fully randomized design, multiple groups of rabbits can be directly
compared. The effective stroke dose or P50 value was then calculated
as the amount of microclots that produce neurological dysfunction
(impairment) in 50% of the rabbits within a specific treatment group.
RLCEM Analyses
Embolized rabbits that died before euthanasia were included in the
study. Their brains were fixed and sectioned as described below.
Surviving animals were euthanized 48 hours after embolization.
Their brains were removed, immersion fixed in 4% paraformalde-
hyde for at least 5 days, and then examined by an observer blinded
to the treatment groups. The right middle cerebral artery also was
examined for the presence of emboli; surface blood vessels were
stripped from the cerebral hemispheres; and the cerebellum was
removed from the brain stem. Hemispheres and brain stem were cut
into seven 5-mm-thick coronal slices, each having 2 faces, for a total
of 14. The presence and size of each hemorrhage and infarct were
noted as described previously.15,18 Hemorrhage size was recorded as
the number of section faces showing hemorrhage, whereas infarction
was grossly visible as pale, softer tissue surrounded by pink, normal
brain tissue on the sections. Finally, the total radioactivity in brain
slices, cerebellum, and brain stem, as well as surface vessels from the
right hemisphere, was measured with a gamma counter. The amount
of radiolabel in the tissues and right hemisphere vessels was
compared with label in the blood clot at embolization. If ⬍10% of
the counts were in the brain and vessels, it was assumed that the
labeled blood clot did not reach the brain,15,18 and data from these
animals were excluded from further analyses.
For all experiments in this study, rabbits were randomly allocated
into treatment groups before the embolization procedure, with
concealment of the randomization guaranteed by the use of an
independent third party. The randomization sequence was not re-
vealed until all postmortem analyses were complete.
Statistical Analysis
For the RSCEM, a separate curve was generated for each treatment
tested. The t test was used for comparison between groups, with the
Bonferroni correction used for multiple comparisons when appropri-
ate. The RLCEM data were analyzed with the ␹2 test for hemor-
rhage/infarct rate and analysis of variance when relevant.
Results
RSCEM
Neuroprotection by Ebselen
Quantal dose-response analysis was used to determine whether
ebselen was neuroprotective after an embolic stroke and whether
 Lapchak and Zivin Synergistic Effect of tPA With Ebselen 2015

TABLE 1. Ebselen and tPA Quantal Analysis: Effects of Ebselen (20 mg/kg) or
tPA (3.3 mg/kg) on Behavioral Outcome of Ischemic Rabbits Measured
Following Embolism
Vehicle Ebselen (5 min) tPA (60 min)

Clot Dose Clot Dose Clot Dose

(mg) Norm Abnorm (mg) Norm Abnorm (mg) Norm Abnorm
1.03 1 0 0.27 1 0 0.55 1 0
1.07 1 0 0.31 1 0 0.56 1 0
1.19 0 1 1.48 1 0 1.35 1 0
1.44 1 0 2.53 0 1 2.28 1 0
1.74 0 0 2.91 0 1 2.31 1 0
1.89 0 1 2.99 1 0 2.56 1 0
2.00 0 1 3.14 1 0 2.85 0 1
2.01 0 1 4.43 0 1 3.29 0 1
2.03 0 1 3.42 1 0
2.18 0 1 3.67 1 0
2.67 1 0 4.04 0 1
2.99 0 1
3.21 0 1
4.36 0 1
4.79 0 1

there was a significant behavioral difference between vehicle-
and ebselen-treated rabbits. Quantal response curves were con-
structed from the raw data presented in Tables 1 and 2 for the
20-mg/kg dose of ebselen and for the vehicle-treated group
(Figure 1). When administered starting 5 minutes after emboli-
zation, ebselen (20 mg/kg) significantly (P⬍0.05) increased the
P50 by 107% compared with the vehicle-treated controls
(1.35⫾0.30 mg) to 2.82⫾0.75 mg, indicating a neuroprotective
effect of the drug at this dose (Figure 1 and Tables 1 and 2). No
significant differences between vehicle and the 10- or 50-mg/kg
doses of ebselen were observed (Figure 2 and Table 3), with P50
values of 2.12⫾0.56 and 0.49⫾0.54 mg, respectively.
Ebselen and tPA Combination
These experiments assessed the effects of treating embo-
lized rabbits with ebselen and tPA. Ebselen (20 mg/kg)

TABLE 2. Ebselen and tPA Quantal Analysis: Effects of Ebselen (20 mg/kg), tPA
(0.9 mg/kg), or Combination Therapy Administered 60 Minutes Following
Embolization on Behavioral Outcome: Quantal Analysis Data
Ebselen tPA Ebselen ⫹ tPA

Clot Dose, Clot Dose, Clot Dose,

mg Norm Abnorm mg Norm Abnorm mg Norm Abnorm
0.04 1 0 0.92 1 0 0.10 1 0
0.09 1 0 1.50 1 0 0.39 1 0
0.59 1 0 1.73 0 1 0.61 1 0
0.86 1 0 1.75 0 1 0.82 1 0
1.56 0 1 1.77 0 1 0.91 1 0
1.59 1 0 2.01 0 1 1.66 1 0
1.66 0 1 2.32 0 1 1.67 1 0
1.82 1 0 2.37 0 1 2.04 1 0
2.07 0 1 2.62 1 0 2.18 1 0
2.19 1 0 2.72 0 1 2.76 0 1
2.36 0 1 2.75 0 1 3.25 1 1
2.59 0 1 2.86 0 1 3.45 1 0
2.91 0 1 3.22 0 1 3.68 1 0
3.52 0 1 3.84 0 1
3.91 0 1
4.12 0 1

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2016 Stroke August 2003

TABLE 3. Effects of Treatments on Behavioral Outcome in

Embolized Rabbits
P50
Drug Treatment and Time (Mean⫾SEM)
5 min postembolization
Vehicle (n⫽16) 1.35⫾0.30
Ebselen (10 mg/kg) (n⫽13) 2.12⫾0.56
Ebselen (20 mg/kg) (n⫽8) 2.82⫾0.75*
Ebselen (50 mg/kg) (n⫽13) 0.49⫾0.54
60 min postembolization
Vehicle (n⫽24) 1.53⫾0.21
Ebselen (20 mg/kg) (n⫽16) 1.69⫾0.32
tPA 3.3 mg/kg (n⫽11) 3.38⫾0.55*
tPA 0.9 mg/kg (n⫽13) 1.54⫾0.36
Figure 1. Percentage of rabbits behaviorally abnormal or dead
Ebselen (20 mg/kg) 5 min⫹tPA (3.3 mg/kg) 60 min (n⫽12) 2.98⫾0.18*
as a function of microclot weight (mg) injected into the carotid
artery system. Dashed curve (left) shows that 50% of vehicle- Ebselen (20 mg/kg) 60 min⫹tPA (3.3 mg/kg) 60 min (n⫽12) 3.60⫾0.79*
treated control rabbits will be abnormal or dead 24 hours after Ebselen (20 mg/kg) 60 min tPA (0.9 mg/kg) 60 min (n⫽16) 3.52⫾0.73*†
injection of 1.35⫾0.30 mg (P50) of clots. Solid curve (right) indi-
cates that when ebselen (20 mg/kg) is given 5 minutes after *P⬍0.05 compared with vehicle; †P⬍0.05 compared with tPA (0.9 mg/kg)
embolization, P50 is significantly increased to 2.82⫾0.75 mg or ebselen (20 mg/kg).
(P⬍0.05).

was given starting 5 or 60 minutes after embolization, with mg/kg) that by itself did not significantly increase the P50
tPA (3.3 mg/kg) injection starting 60 minutes after embo- value (1.54⫾0.35 mg) was used in combination with
lization. With tPA alone, a significant increase in the P50 ebselen (20 mg/kg) and given 60 minutes after emboliza-
(P50⫽3.38⫾0.55 mg) was noted. When the 2 drugs were tion, which also did not significantly increase the P50 value
administered in combination, there was no additional
(1.65⫾0.32 mg). A statistically significant increase
behavioral improvement: the P50 values for the combina-
(P⬍0.05) in the P50 value (3.52⫾0.73 mg) was observed
tion groups were 2.98⫾0.18 and 3.60⫾0.79 mg for the 5-
with this combination (Table 3 and Figure 3), suggesting a
and 60-minute ebselen treatment, respectively (Table 3).
synergistic effect of the drugs on behavioral improvement.
That the combination did not significantly increase P50
values compared with monotherapy suggests that a ceiling
effect caused by the high dose of tPA and/or the short
delay (5 minutes) between embolization and ebselen treat-
ment may have occurred but does not exclude the possi-
bility of a positive result if a lower dose of tPA and a
longer delay (60 minutes) for ebselen treatment were used.
To test this hypothesis, a suboptimal dose of tPA (0.9

Figure 3. Percentage of rabbits behaviorally abnormal or dead

Figure 2. Dose-response analysis for the effect of ebselen (10 to
50 mg/kg) on behavioral outcome (P50) measured after embo-
lism. Ebselen (20 mg/kg) increased P50 (P⬍0.05), but other
doses did not (P⬎0.05).
as a function of microclot weight (mg) injected into the carotid
artery system. Drugs were given 60 minutes after embolization.
Dashed curve (left) shows that 50% of vehicle-treated control
rabbits treated with 1.53⫾0.21 mg clots (P50) will be abnormal
or dead 24 hours after injection. Solid curve overlapping the
vehicle curve shows that tPA (0.9 mg/kg) administered 60 min-
utes after embolization results in P50 of 1.54⫾0.36 mg (P⬎0.05).
Dotted/dashed curve shows that ebselen (20 mg/kg) is ineffec-
tive when administered 60 minutes after embolization, but when
combined with tPA (0.9 mg/kg) also given 60 minutes after
embolization (far right curve), the P50 value is significantly
increased to 3.52⫾0.73 mg (P⬍0.05).

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 Lapchak and Zivin
RLCEM
Hemorrhage and Infarct Incidence
The 2 doses of tPA (0.9 and 3.3 mg/kg) used in the RSCEM
experiments were evaluated for effects on hemorrhage rate in
the RLCEM. Hemorrhage rate was 50% (5 of 10), 64% (9 of
14), and 76% (13 of 17) for the vehicle- and 0.9- and
3.3-mg/kg-tPA–treated groups, respectively.
Discussion
Antioxidants and free radical scavengers have been devel-
oped as therapeutics for the treatment of ischemic stroke for
many years.9 However, because to date all attempts at
neuroprotection with single compounds have been unsucces-
ful,12,19,20 it is likely that combination therapies such as a
thrombolytic with a neuroprotective small molecule will be
necessary to achieve optimal neuroprotection and clinical
improvements after an ischemic stroke.13,14,16,20 –24 Thrombo-
lytics are valuable agents because recanalization allows not
only reperfusion of ischemic tissue when the stroke is the
result of an embolus but also access of small-molecule drugs
to the penumbra of the infarcted tissue.21,25,26
In the present study, we assessed the pharmacological
effects of ebselen, an anti-inflammatory antioxidant that
mimics glutathione peroxidase activities,1 in the absence or
presence of recanalization such as with tPA. The results in the
RSCEM showed that ebselen significantly improved behav-
ioral ratings scores after an embolic stroke, but only when
administered relatively soon (5 minutes) after embolization.
The observation that ebselen is neuroprotective in the RS-
CEM is consistent with earlier findings in rodent stroke
models.6,27–30 However, with the highest dose tested, ebselen
did not significantly affect the P50 compared with controls,
perhaps because of the detrimental effects mediated by its
activity on many enzymes and proteins.1 Nevertheless, the
neuroprotective properties of ebselen in the RSCEM are
comparable to those of the spin-trap agent NXY-059 in the
same model.9,16 Therefore, it appears that intervention at the
level of oxidative stress, reactive oxygen species, nitrogen
reactive species, and free radicals is an effective way to
produce neuroprotection and significantly improve behavior
in the RSCEM.
Delayed administration of ebselen in combination with a
low dose of the thrombolytic tPA presented a superior
pharmacological profile compared with either drug alone.
The synergism between ebselen and tPA indicates that it may
be possible to administer a lower dose of thrombolytic,
thereby reducing complications associated with thrombolytic
therapy (eg, hemorrhage21) while still providing maximal
behavioral improvement. This is supported by the RLCEM
studies showing that a low dose of tPA (0.9 mg/kg) increased
hemorrhage rate by only 28% compared with the high dose
(3.3 mg/kg), which increased the rate by 52%. Alternatively,
ebselen may reduce reperfusion-induced injury caused by
tPA and consequently protect affected neurons. Previous
studies using rabbit embolic stroke models have shown that
tPA effectively lyses blood clots and quickly restores cerebral
reperfusion31 (reviewed elsewhere21).
The present preclinical data, together with the results from
ebselen clinical trials, imply that ultimately the development
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Synergistic Effect of tPA With Ebselen 2017
of combination drug therapy may be necessary to reduce the
devastating neurological and behavioral deficits of acute
ischemic stroke. Because of the observed synergistic effects
of ebselen with tPA, ebselen or other neuroprotective small-
molecules like NXY-0599 may be prime candidates for
combination therapy with tPA or second-generation
thrombolytics like Tenecteplase or microplasmin.15,21 Future
placebo-controlled, double-blind clinical trials are needed to
test the hypothesis that combination therapies will result in
better outcomes for stroke patients.
Conclusions
Ebselen is neuroprotective because it significantly improved
behavioral rating scores in a rabbit embolic stroke model. The
limited dose and time of administration range indicate that
ebselen has a narrow therapeutic window. However, our
results showing a synergistic effect of ebselen plus tPA
indicate that ebselen may be administered concomitantly with
lower-dose thrombolytics to produce significant neuroprotec-
tion and behavioral improvements while reducing the inci-
dence of thrombolytic side effects (ie, hemorrhage rate).
Acknowledgments
This study was supported by funding from NS28121 (P.A.L./J.A.Z.),
NS23814 (J.A.Z.), VA Merit Review (J.A.Z.), and the Christopher
Reeve Paralysis Foundation (P.A.L.). We thank Dr Dalia M. Araujo
for critical comments, Donghuan Song and Jiandong Wei for
technical assistance, and Genentech Inc for supplying tPA.
References
1. Schewe T. Molecular actions of ebselen: an antiinflammatory antioxidant.
Gen Pharmacol. 1995;26:1153–1169.
2. Herin GA, Du S, Aizenman E. The neuroprotective agent ebselen
modifies NMDA receptor function via the redox modulatory site. J Neu-
rochem. 2001;78:1307–1314.
3. Bosch-Morell F, Roma J, Marin N, Romero B, Rodriguez-Galietero A,
Johnsen-Soriano S, Diaz-Llopis M, Romero FJ. Role of oxygen and
nitrogen species in experimental uveitis: anti- inflammatory activity of
the synthetic antioxidant ebselen. Free Radic Biol Med. 2002;33:
669 – 675.
4. Possel H, Noack H, Keilhoff G, Wolf G. Life imaging of peroxynitrite in
rat microglial and astroglial cells: role of superoxide and antioxidants.
Glia. 2002;38:339 –350.
5. Bailly F, Zoete V, Vamecq J, Catteau JP, Bernier JL. Antioxidant actions
of ovothiol-derived 4-mercaptoimidazoles: glutathione peroxidase
activity and protection against peroxynitrite-induced damage. FEBS Lett.
2000;486:19 –22.
6. Namura S, Nagata I, Takami S, Masayasu H, Kikuchi H. Ebselen reduces
cytochrome c release from mitochondria and subsequent DNA fragmen-
tation after transient focal cerebral ischemia in mice. Stroke. 2001;32:
1906 –1911.
7. Rossato JI, Zeni G, Mello CF, Rubin MA, Rocha JB. Ebselen blocks the
quinolinic acid-induced production of thiobarbituric acid reactive species
but does not prevent the behavioral alterations produced by intra-striatal
quinolinic acid administration in the rat. Neurosci Lett. 2002;318:
137–140.
8. Porciuncula LO, Rocha JB, Boeck CR, Vendite D, Souza DO. Ebselen
prevents excitotoxicity provoked by glutamate in rat cerebellar granule
neurons. Neurosci Lett. 2001;299:217–220.
9. Lapchak PA. NXY-059 evaluation. Curr Opin Invest Drugs. 2002;3:
1758 –1762.
10. Yamaguchi T, Sano K, Takakura K, Saito I, Shinohara Y, Asano T,
Yasuhara H. Ebselen in acute ischemic stroke: a placebo-controlled,
double-blind clinical trial: Ebselen Study Group. Stroke. 1998;29:12–17.
11. Ogawa A, Yoshimoto T, Kikuchi H, Sano K, Saito I, Yamaguchi T,
Yasuhara H. Ebselen in acute middle cerebral artery occlusion: a placebo-
controlled, double-blind clinical trial. Cerebrovasc Dis. 1999;9:112–118.
2018 Stroke August 2003
12. Muir KW. Heterogeneity of stroke pathophysiology and neuroprotective
clinical trial design. Stroke. 2002;33:1545–15450.
13. Recommendations for standards regarding preclinical neuroprotective
and restorative drug development. Stroke. 1999;30:2752–2758.
14. Recommendations for clinical trial evaluation of acute stroke therapies.
Stroke. 2001;32:1598 –1606.
15. Lapchak PA, Araujo DM, Pakola S, Song D, Wei J, Zivin JA. Micro-
plasmin: a novel thrombolytic that improves behavioral outcome after
embolic strokes in rabbits. Stroke. 2002;33:2279 –2284.
16. Lapchak PA, Araujo DM, Song D, Wei J, Zivin JA. Neuroprotective effects
of the spin trap agent disodium-[(tert-butylimino)methyl]benzene-1,
3-disulfonate N-oxide (generic NXY-059) in a rabbit small clot embolic
stroke model: combination studies with the thrombolytic tissue plasminogen
activator. Stroke. 2002;33:1411–1415.
17. Zivin JA, Waud DR. Quantal bioassay and stroke. Stroke. 1992;23:
767–773.
18. Lapchak PA, Araujo DM, Song D, Wei J, Purdy R, Zivin JA. Effects of
the spin trap agent disodium-[tert-butylimino)methyl]benzene-1,
3-disulfonate N-oxide (generic NXY-059) on intracerebral hemorrhage in
a rabbit large clot embolic stroke model: combination studies with tissue
plasminogen activator. Stroke. 2002;33:1665–1670.
19. Tirilazad mesylate in acute ischemic stroke: a systematic review:
Tirilazad International Steering Committee. Stroke. 2000;31:2257–2265.
20. Muir KW, Grosset DG. Neuroprotection for acute stroke: making clinical
trials work. Stroke. 1999;30:180 –182.
21. Lapchak PA. Development of thrombolytic therapy for stroke: a per-
spective. Expert Opin Invest Drugs. 2002;11:1623–1632.
Downloaded from http://stroke.ahajournals.org/ by guest on November 14, 2015
22. Tanahashi N, Fukuuchi Y. Treatment of acute ischemic stroke: recent
progress. Intern Med. 2002;41:337–344.
23. Heiss WD. Stroke: acute interventions. J Neural Transm Suppl. 2002;63:
37–57.
24. Ovbiagele B, Kidwell CS, Starkman S, Saver JL. Neuroprotective agents
for the treatment of acute ischemic stroke. Curr Neurol Neurosci Rep.
2003;3:9 –20.
25. Zivin JA. Thrombolytic stroke therapy: past, present, and future. Neu-
rology. 1999;53:14 –19.
26. Lapchak PA. Hemorrhagic transformation following ischemic stroke:
significance, causes, and relationship to therapy and treatment. Curr
Neurol Neurosci Rep. 2002;2:38 – 43.
27. Imai H, Masayasu H, Dewar D, Graham DI, Macrae IM. Ebselen protects
both gray and white matter in a rodent model of focal cerebral ischemia.
Stroke. 2001;32:2149 –2154.
28. Kondoh S, Nagasawa S, Kawanishi M, Yamaguchi K, Kajimoto S, Ohta
T. Effects of ebselen on cerebral ischemia and reperfusion evaluated by
microdialysis. Neurol Res. 1999;21:682– 686.
29. Takasago T, Peters EE, Graham DI, Masayasu H, Macrae IM. Neuropro-
tective efficacy of ebselen, an anti-oxidant with anti-inflammatory
actions, in a rodent model of permanent middle cerebral artery occlusion.
Br J Pharmacol. 1997;122:1251–1256.
30. Dawson DA, Masayasu H, Graham DI, Macrae IM. The neuroprotective
efficacy of ebselen (a glutathione peroxidase mimic) on brain damage
induced by transient focal cerebral ischaemia in the rat. Neurosci Lett.
1995;185:65– 69.
31. Russell D, Madden KP, Clark WM, Zivin JA. Tissue plasminogen acti-
vator cerebrovascular thrombolysis in rabbits is dependent on the rate and
route of administration. Stroke. 1992;23:388 –393.
 Ebselen, a Seleno-Organic Antioxidant, Is Neuroprotective After Embolic Strokes in
Rabbits: Synergism With Low-Dose Tissue Plasminogen Activator
Paul A. Lapchak and Justin A. Zivin

Stroke. 2003;34:2013-2018; originally published online July 10, 2003;

doi: 10.1161/01.STR.0000081223.74129.04
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