Natural Vitamin E ␣-Tocotrienol Protects Against Ischemic
Stroke by Induction of Multidrug Resistance-Associated
Protein 1
Han-A Park, PhD; Natalia Kubicki, BS; Surya Gnyawali, PhD; Yuk Cheung Chan, PhD;
Sashwati Roy, PhD; Savita Khanna, PhD; Chandan K. Sen, PhD
Background and Purpose—␣-Tocotrienol (TCT) represents the most potent neuroprotective form of natural vitamin E that
is Generally Recognized As Safe certified by the U.S. Food and Drug Administration. This work addresses a novel
molecular mechanism by which ␣-TCT may be protective against stroke in vivo. Elevation of intracellular oxidized
glutathione (GSSG) triggers neural cell death. Multidrug resistance-associated protein 1 (MRP1), a key mediator of
intracellular oxidized glutathione efflux from neural cells, may therefore possess neuroprotective functions.
Methods—Stroke-dependent brain tissue damage was studied in MRP1-deficient mice and ␣-TCT-supplemented mice.
Results—Elevated MRP1 expression was observed in glutamate-challenged primary cortical neuronal cells and in
stroke-affected brain tissue. MRP1-deficient mice displayed larger stroke-induced lesions, recognizing a protective role
of MRP1. In vitro, protection against glutamate-induced neurotoxicity by ␣-TCT was attenuated under conditions of
MRP1 knockdown; this suggests the role of MRP1 in ␣-TCT-dependent neuroprotection. In vivo studies demonstrated
that oral supplementation of ␣-TCT protected against murine stroke. MRP1 expression was elevated in the
stroke-affected cortical tissue of ␣-TCT-supplemented mice. Efforts to elucidate the underlying mechanism identified
MRP1 as a target of microRNA (miR)-199a-5p. In ␣-TCT-supplemented mice, miR-199a-5p was downregulated in
stroke-affected brain tissue.
Conclusions—This work recognizes MRP1 as a protective factor against stroke. Furthermore, findings of this study add
a new dimension to the current understanding of the molecular bases of ␣-TCT neuroprotection in 2 ways: by identifying
MRP1 as a ␣-TCT-sensitive target and by unveiling the general prospect that oral ␣-TCT may regulate miR expression
in stroke-affected brain tissue. (Stroke. 2011;42:2308-2314.)
Key Words: antioxidant Ⅲ vitamin E Ⅲ microRNA Ⅲ glutathione
Vitamin E is a generic term for tocopherols (TCP) and
tocotrienols (TCT). Although TCPs have been widely
studied for decades, the significance of ␣-TCT as the most
potent neuroprotective form of natural vitamin E has been
uncovered recently. We have reported that nanomolar
concentrations of ␣-TCT, not ␣-TCP, prevent strokeassociated
neurodegeneration.1–2 Alpha-TCT protects neural
cells by 2 major mechanisms, including inhibition of
inducible c-Src and 12-lipoxygenase pathways.1,3 Recently,
we have reported the key role of intracellular
oxidized glutathione (GSSG) in causing neural cell death.4
Under normal physiological conditions, in most tissues
GSSG represents 1% of total glutathione in the cell.
However, under conditions of oxidative stress, reduced
glutathione (GSH) is rapidly oxidized to GSSG. Once
intracellular GSSG is formed, it may be recycled to GSH
in the presence of reducing equivalents, which are depleted
in the face of oxidant insult. Excessive cellular GSSG is
toxic and therefore is pumped out at the expense of
adenosine triphosphate. We have demonstrated that inefficient
clearance of intracellular GSSG may trigger neural
cell death. These observations highlight the significance of
cellular GSSG-clearing systems in neuroprotection, especially
in the context of stroke, where oxidative stress is
overt.4
Multidrug resistance-associated protein 1 (MRP1) plays a
key role in clearing intracellular GSSG.5 MRP1 is an integral
membrane glycophosphoprotein abundantly expressed in the
brain. We sought to test the hypothesis that under conditions
of oxidant insult, improved clearance of intracellular GSSG
by augmented MRP1 function results in neuroprotective
outcomes. Furthermore, this study tested orally supplemented
natural vitamin E ␣-TCT for its ability to enhance MRP1
function in the context of stroke-induced brain injury.
Received November 15, 2010; accepted February 25, 2011.
From the Department of Surgery, Davis Heart and Lung Research Institute, Ohio State University Medical Center, Columbus, Ohio 43210.
The online-only Data Supplement is available at http://stroke.ahajournals.org/cgi/content/full/STROKEAHA.110.608547/DC1.
Correspondence to Chandan K. Sen, 512 Davis Heart & Lung Research Institute, 473 West 12th Avenue, Ohio State University Medical Center,
Columbus, Ohio 43210. E-mail chandan.sen@osumc.edu
© 2011 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.110.608547
2308
Materials and Methods
For specific details, see online supplement at http://aha.strokejournal.org.
Primary Cortical Neurons
Cells were isolated from the cerebral cortex of rat feti (SpragueDawley,
day 17 of gestation; Harlan; see online supplement at
http://aha.strokejournal.org).
siRNA Delivery and Analysis of Genes
DharmaFECT 1 transfection reagent was used to transfect cells with
100nmol/L siRNA pool (Dharmacon RNA Technologies; see online
supplement at http://aha.strokejournal.org).
Delivery of miR Mimic and Inhibitor
DharmaFECT 1 transfection reagent was used to transfect cells with
miRIDIAN rno-miR-199a-5p mimic or inhibitor (Dharmacon RNA
Technologies; see online supplement at http://aha.strokejournal.org).
pGL3-MRP1–3؅ Untranslated Region Luciferase
Reporter Assay
See online supplement at http://aha.strokejournal.org
Quantification of miR Expression
miR fraction was isolated using miRVana miRNA isolation kit
(Ambion). miR-199a-5p levels were quantified using Taqman Universal
Master Mix (Applied Biosystems; see online supplement at
http://aha.strokejournal.org).
Western Blot
Samples (40–50 ␮g of protein/lane) were separated on a 4% to 12%
sodium dodecyl sulfate polyacrylamide and probed with anti-MRP1
(1:20 dilution, Enzo Life Sciences; see online supplement at
http://aha.strokejournal.org).
Cell Viability Assay
See online supplement at http://aha.strokejournal.org.
Immunocytochemistry
See online supplement at http://aha.strokejournal.org.
Calcein Clearance Assay
After MRP1 siRNA transfection, primary cortical neurons were
treated with glutamate. Calcein-AM (5 ␮mol/L, Invitrogen) was
loaded to the cells for 30 minutes, and loss of cellular fluorescence
was analyzed (see online supplement at http://aha.strokejournal.org).
Figure 1. MRP1 knockdown attenuated neuroprotection of ␣-tocotrienol in primary cortical neurons. After 24 hours of seeding, primary
cortical neurons were treated with glutamate (5mmol/L, 24 hours). Glutamate challenge induced the expression of MRP1 protein (A).
After MRP1 siRNA transfection, MRP1 mRNA expression was significantly downregulated (B). C, Glutamate-challenged primary neurons
with MRP1 knockdown exhibited loss of functional MRP1 by retaining more calcein compared with corresponding control cells. D,
␣-TCT (1␮mol/L) was added into cell culture medium 6 hours before glutamate treatment. After glutamate (5mmol/L, 24 hours) challenge,
LDH leakage was measured. Neuroprotection of ␣-TCT was compromised under conditions of MRP1 knockdown. Barϭ50 ␮m,
nϭ3, *PϽ0.05 compared with control; §PϽ0.05 compared with control siRNA-transfected, glutamate-treated primary neurons. SiRNA
indicates small interfering RNA; mRNA, messenger RNA; TCT, Tocotrienol; LDH, lactate dehydrogenase.
Park et al Vitamin E ␣-Tocotrienol Against Stroke 2309
Mouse Stroke Model
Transient focal cerebral ischemia was induced in 8-week-old, MRP1deficient
(nϭ20, male; Taconic) or background friend virus B-type
(FVB) mice (nϭ15, male; Taconic) by middle cerebral artery occlusion
(MCAO; see online supplement at http://aha.strokejournal.org).
Magnetic Resonance Imaging
T2-weighted imaging was performed on stroke-affected mice. Imaging
experiments were carried out using an 11.7 T magnetic resonance imaging
system comprised of a vertical bore magnet (Bruker Biospin; see online
supplement at http://aha.strokejournal.org).
Figure 2. MCAO-induced brain injury was
exacerbated in MRP1-deficient mice.
Transient focal cerebral ischemia was
induced in MRP1-deficient (nϭ20) or
friend virus B-type (FVB) mice (nϭ15) by
MCAO. A, MRP1 KO mice presented
larger stroke-induced lesion compared
with FVB mice after 48 hours reperfusion
(FVB, nϭ8; MRP1 KO, nϭ12). B, Protein
expression of MRP1 was increased in
infarct hemisphere of FVB mice (nϭ3). C,
MRP1-deficient mice contained significantly
higher level of GSSG in strokeaffected
hemisphere compared with that
of FVB mice (nϭ4). *PϽ0.05 compared
with corresponding contralateral hemisphere;
†PϽ0.05 compared with FVB;
§PϽ0.05 compared with infarct hemisphere
of FVB. MCAO indicates middle
cerebral artery occlusion; KO, knockout;
GSSG, intracellular oxidized glutathione.
Figure 3. MCAO-induced neurodegeneration was accentuated in MRP1-deficient mice. A, MRP1 expression was upregulated at the
stroke-affected cortex of friend virus B-type (FVB) mice (red-MRP1 protein; blue-DAPI stained nuclei) B, The infarct hemisphere of
MRP1 deficient mice presented abundant Fluoro-Jade signals in cortical region (green-Fluoro-Jade positive neurons; blue-DAPI stained
nuclei). Barϭ50 ␮m, nϭ3, *PϽ0.05 compared with corresponding contralateral hemisphere; §PϽ0.05 compared with infarct hemisphere
of FVB. MCAO indicates middle cerebral artery occlusion.
2310 Stroke August 2011
␣-Tocotrienol Supplementation
C57BL/6 (5-week old, male; Harlan) mice were randomly divided
into 2 groups: control (nϭ18) and supplemented (nϭ23). The control
group was orally gavaged with vitamin-E-stripped corn oil, with
volume matching the mean volume of ␣-TCT supplement group. The
test group was orally gavaged with ␣-TCT (Carotech) in vitamin-Estripped
corn oil at a dosage of 50 mg/kg body weight for 13 weeks
as reported previously.1 MCAO was performed at 20 to 24 hours
after the last supplementation. After 48 hours of MCAO, T2weighted
images were recorded. Mice suffering from surgical
complications (eg, hemorrhage or death) during MCAO were excluded.
Immediately after imaging, tissues from control and strokeaffected
hemispheres of control (nϭ9) and ␣-TCT-supplemented
(nϭ10) mice were harvested. Mice were maintained under standard
conditions at 22Ϯ2°C with 12:12 dark:light cycles. All animal
protocols were approved by the Institutional Animal Care and Use
Committee of Ohio State University.
HPLC-Electrochemical Detection
See online supplement at http://aha.strokejournal.org.
mRNA Expression Assay From Laser-Captured
Microdissected Somatosensory Cortex of
Brain Sample
Laser microdissection and pressure catapulting was performed on
coronal slices of mouse brains using the microlaser system from
PALM Microlaser Technologies AG. (See online supplement at
http://aha.strokejournal.org).
Histology
Coronal slices of cortical sections were stained with rabbit polyclonal
antibody to MRP1 (1:200, Abbiotec), 0.0001% Fluoro-Jade C
(Millipore), or 4-hydroxynonenal (1:1000; Enzo Life Sciences; see
online supplement at http://aha.strokejournal.org).
Statistics
Data are reported as meanϮSD. Differences between means were
tested using Student t test or 1-way ANOVA with Tukey’s test.
PϽ0.05 was considered statistically significant.
Results
Neuroprotection of ␣-Tocotrienol was
Compromised in MRP1 Knockdown in Primary
Cortical Neurons
In neural cells, glutamate toxicity is associated with oxidant
insult, resulting in rapid elevation of intracellular GSSG. We
were therefore led to test the hypothesis that under such
conditions of GSSG loading, GSSG efflux mechanisms such
as MRP1 would be augmented as a defense response in favor
of neuronal survival. We observed that extracellular glutamate
challenge may induce MRP1 expression in both primary
cortical neurons (Figure 1A) as well as in HT4 neural cells
(Figure S1A).
Previously we have reported that ␣-TCT is potently neuroprotective
against a number of insults, including GSSGinduced
cell death.3–4,6 To test whether such neuroprotective
property of ␣-TCT depends on MRP1 in neural cells, cellular
GSSG clearance was studied utilizing a MRP1 knockdown
approach (Figure 1B and Figure S1B-D). Higher MRP1
activity resulted in enhanced clearance of calcein from
preloaded neurons. Glutamate challenge upregulated MRP1
function. Such improved clearance of calcein in glutamatechallenged
cells was blunted by MRP1 knockdown demonstrating
specificity of the assay for MRP1 (Figure 1C). The
ability of ␣-TCT to protect against glutamate challenge was
compromised following MRP1 knockdown in primary cortical
neurons (Figure 1D).
MCAO-Induced Brain Injury Was Exacerbated in
MRP1-Deficient Mice
Transient focal cerebral ischemia was induced by MCAO in
MRP1-deficient mice or corresponding background FVB
mice. Both FVB and MRP1-deficient mice showed comparably
decreased levels of middle cerebral artery–area blood
flow during occlusion (not shown). Interestingly, MRP1deficient
mice showed a larger hemispherical infarct volume
than that noted in FVB mice (Figure 2A). Increased abundance
of MRP1 protein was observed in the infarct hemisphere
of FVB mice (Figure 2B). MCAO-induced brain
injury increased tissue GSSG levels in both MRP1-deficient
as well as in FVB mice. Notably, MRP1-deficient mice
contained 1.6-fold higher tissue levels of GSSG in the infarct
hemisphere compared with that detected in background mice
(Figure 2C). These data indicate that loss of MRP1 function
impairs GSSG clearance during stroke, resulting in increased
brain injury. Histochemical studies depicted that increased
MRP1-positive cells were selectively localized in the infarct
hemisphere of FVB mice (Figure 3A). Both MRP1-deficient,
as well as FVB, mice presented positive Fluoro-Jade immunofluorescence
staining in the stroke-affected cortex, supportFigure
4. miR-199a–5p silences MRP1 in primary cortical neurons.
After 72 hours of miR-199a–5p mimic delivery, miR-199a–5p
expression was significantly upregulated (A) while miR-199a–5p
hairpin inhibitor significantly downregulated miR-199a–5p (B). miR-
199a–5p mimic/inhibitor negatively regulated protein (C-D) of
MRP1 in primary cortical neurons. nϭ3, *PϽ0.05 compared with
control.
Park et al Vitamin E ␣-Tocotrienol Against Stroke 2311
ing the incidence of neurodegeneration. Importantly, the
infarct hemisphere of MRP1-deficient mice exhibited a
higher level of neurodegenerative outcome (Figure 3B).
miR-199a-5p Silences MRP1
To test whether MRP1 expression is subject to posttranscriptional
gene silencing by miR, we first performed in
silico studies using databases such as Target Scan (www.targetscan.org),
Miranda (www.microrna.org), and PicTar
(www.pictar.org). miR-199a-5p emerged as a major candidate
miR, which is likely to target MRP1 exhibiting a single
conserved binding site to the 3Ј untranslated region of MRP1
transcript. Next, we turned toward biological validation of
MRP1 as a target of miR-199a-5p. In cultured cells, miR-
199a-5p levels were modulated by delivery of miR-199a-5p
mimic or miR-199a-5p inhibitor (Figure 4A-B and Figure
S2A). miR-199a-5p silenced MRP1 in both primary (Figure
4C-D) as well as in HT4 cells (Figure S2B-C). To determine
whether MRP1 is a direct target of miR-199a-5p in HT4
neural cells, a fragment of the 3Ј untranslated region of MRP1
mRNA containing the putative miR-199a-5p binding sequence
cloned into a firefly luciferase reporter construct was
used. This construct was cotransfected with a control renilla
luciferase reporter construct into cells along with miR-
199a-5p mimic or inhibitor. miR-199a-5p mimic significantly
decreased luciferase activity, while miR-199a-5p inhibitor
significantly increased the activity of the miR-199a-5p firefly
luciferase reporter; this finding recognizes MRP1 as a direct
target of miR-199a-5p (Figure S2D).
Oral ␣-Tocotrienol Protected Against Stroke via
MRP1 Upregulation
To test the neuroprotective effects of ␣-TCT against stroke in
vivo, randomly divided mice were gavaged with either
vitamin-E-stripped corn oil or ␣-TCT (50 mg/kg body
weight) for 13 weeks. MCAO was performed 1 day after the
last supplementation. MCAO-affected brains were harvested
48 hours after reperfusion (Figure S3A). Alpha-TCT supplementation
significantly increased brain ␣-TCT level (Figure
S3B) without changing ␣-TCP levels (Figure S3C). MCAO
limited blood flow in ␣-TCT-supplemented, as well as in
control groups, comparably (Figure S3D). However, MCAOinduced
hemispherical infarct volume was significantly attenuated
in ␣-TCT-supplemented group (Figure 5A).
To test whether the neuroprotective properties of ␣-TCT
against stroke in vivo was dependent on the ability of ␣-TCT
to upregulate MRP1, cortical tissue elements were microdissected
from infarct, as well as contralateral noninfarct, sites
using a laser microdissection and pressure catapulting system
(Figure S4). Notably, ␣-TCT supplementation induced
MRP1, but lowered miR199a-5p level, in the infarct hemisphere
(Figure 5B-D). Immunohistochemical studies demonstrated
higher abundance of MRP1-positive cells at the infarct
hemisphere of ␣-TCT-supplemented mice as compared to
corn oil supplemented mice (Figure 6A). Alpha-TCT supplementation
attenuated stroke-induced neurodegeneration (Figure
6B). Stroke induced lipid peroxidation, as evidenced by
elevated levels of 4-hydroxynonenal–positive cells, in the
infarct tissue. Consistent with other observations indicating a
beneficial influence of ␣-TCT supplementation, 4-hydroxynonenal–positive
cells were fewer in the infarct hemisphere
of ␣-TCT-supplemented mice (Figure 6C).
Discussion
Following a number of failed clinical trials testing the
conventional form of vitamin E, ␣-TCP,7–8 interest in naturally
occurring forms of vitamin E such as ␥-TCP, as well as
the TCT family, is sharply rising. Meta-analyses of clinical
trials testing the efficacy of vitamin E in human health suffer
from a blind spot because they fail to recognize that ␣-TCP,
Figure 5. Orally supplemented
␣-tocotrienol protected against stroke via
MRP1 upregulation. C57BL/6 mice were
orally gavaged with vitamin-E-stripped
corn oil (nϭ18) or 50 mg ␣-TCT/kg body
weight (nϭ23) for 13 weeks, then MCAO
was performed. A, ␣-TCT supplementation
significantly reduced MCAO-induced
brain injury (corn-oil-supplemented mice,
nϭ9; ␣-TCT supplemented mice, nϭ10).
miR-199a–5p expression was significantly
downregulated (B) while MRP1 mRNA (C)
and protein (D) levels were significantly
elevated in the stroke-affected tissue of
␣-TCT-fed mice (nϭ3). *PϽ0.05 compared
with corresponding control;
§PϽ0.05 compared with infarct hemisphere
of corn-oil-fed mice.
2312 Stroke August 2011
the only form of vitamin E tested in such trials, represents
only a fraction of the natural vitamin E family.7–8 Because
TCPs and TCTs have unique functional properties, it is
important to limit title claims to the specific form of vitamin
E studied. Neuroprotection by ␣-TCT at nanomolar concentration
represents the most potent functional property of the
entire vitamin E family.6 Mechanisms explaining such property
include inhibition of inducible c-Src, as well as 12-lipoxygenase
activity, in stroke-related neurodegenerative
settings.1,9
Stroke-associated ischemic insult is known to compromise
adenosine triphosphate production as well as deplete the
reducing equivalent pool of the brain. This, in turn, compromises
the function of adenosine-triphosphate-dependent ion
pumps, as well as the ability of the brain to manage oxidant
insult. As a result, Kϩ
efflux and Ca2ϩ
influx are increased in
response to membrane depolarization. In contrast, excessive
GSSG builds up within the cell. Both elevated intracellular
Ca2ϩ
, as well as GSSG, are directly implicated in cell death
signaling.10–11 Our previous work highlights the critical
significance of elevated and trapped cellular GSSG in executing
neural cell death.4
MRP1 represents a major GSSG clearing system in neural
cells.12 The current work is the first to recognize MRP1 as a
potential therapeutic target for stroke. During stroke-related
oxidative insult, GSSG is rapidly formed, but its redox
cycling to GSH is severely arrested. GSSG reductase activity
is impaired following stroke.13 In addition, ischemiareperfusion
depletes nioctinamide dinucleotide phosphate,
impairing all reductase functions dependent on this reducing
equivalent.14 Therefore, a sharp rise in brain tissue GSSG/
GSH ratio occurs following stroke.4 In the current article, we
note that such elevation of intracellular GSSG is associated
with induction of MRP1, perhaps as an adaptive survival
response. This contention is consistent with previous reports
demonstrating upregulation of MRP1 in response to oxidant
insult as an adaptive response defending cell survival.12
Mechanisms of multidrug resistance and clinical outcomes in
Figure 6. Orally supplemented ␣-tocotrienol attenuated MCAO-induced neurodegeneration and lipid peroxidation. A, The abundance of
MRP1-positive cells were significantly upregulated in the stroke-affected cortex of ␣-TCT-supplemented mice (red-MRP1 protein; blueDAPI
stained nuclei). B, Fluoro-Jade-positive neurons were fewer in the infarct hemisphere of ␣-TCT-fed group compared with those at
the infarct site of corn-oil-fed group (green-Fluoro-Jade; blue-DAPI stained nuclei). C, The abundance of 4-HNE–positive cells was
lower in infarct hemisphere of ␣-TCT-supplemented mice. Bar (white)ϭ50 ␮m, Bar (black)ϭ20 ␮m. nϭ3, *PϽ0.05 compared with corresponding
contralateral hemisphere; §PϽ0.05 compared with infarct hemisphere of corn oil fed mice. MCAO indicates middle cerebral
artery occlusion; MRP1, multidrug resistance-associated protein 1; TCT, tocotrienol; DAPI, 4=,6-diamidino-2-phenylindole; HNE,
hydroxynonenal.
Park et al Vitamin E ␣-Tocotrienol Against Stroke 2313
response to manipulation of MRP1 expression have been
extensively studied in the context of various types of cancers.
However, the significance of MRP1 in brain-related pathologies
other than cancer is poorly developed. Currently, the
limited information available consistently demonstrates that
under pathological conditions known to be associated with
oxidant insult, ie, Alzheimer’s disease and epilepsy, MRP1
expression is elevated in the brain.15–16 Our observation that
stroke-related injury to the brain is exacerbated in MRP1deficient
mice establishes a key significance of MRP1 in
determining stroke outcomes.
The current study identified MRP1 as a biologically
validated target of miR-199a-5p. This constitutes the first
evidence that MRP1 is subject to post-transcriptional gene
silencing by miRs. This finding provided a great segue into
addressing the significance of miRs in stroke.17 Antagomir
miR-497 treatment has been recognized for its potential to
attenuate injury related to acute ischemic stroke.18 The
miR-200 family and miR-182 have been found to be upregulated
in ischemic preconditioning.19 Although the significance
of miR-199a-5p in stroke has not been studied, miR-
199a-5p is recognized as a hypoxia-sensitive miR.20 Thus, the
hypoxia component of stroke-related ischemia may be responsible
for downregulating miR-199a-5p following
stroke.21
Conclusions
The findings of this study add a new dimension to the current
understanding of the molecular bases of ␣-TCT neuroprotection
in 2 ways: by identifying MRP1 as a ␣-TCT-sensitive
target and by unveiling the general prospect that oral ␣-TCT
may regulate microRNA expression in stroke-affected brain
tissue. Neuroprotective, as well as hypocholesterolemic,
properties of ␣-TCT make it a good candidate for nutritionbased
intervention in people at high risk for stroke. Transient
ischemic attack, or mini-stroke, serves as a sentinel warning
sign for high-risk stroke patients.22 Prophylactic stroke therapy
therefore provides an opportunity for intervention in
patients experiencing transient ischemic attack before a major
stroke event. Outcomes of the current study warrant clinical
assessment of ␣-TCT in transient ischemic attack patients.
Furthermore, ␣-TCT is a nutrient that is certified by the U.S.
Food and Drug Administration to be Generally Recognized
As Safe (GRN307) and is not a drug with potential side
effects. Thus, ␣-TCT may be considered as a preventive
nutritional countermeasure for people at high risk for stroke.
Sources of Funding
This study was supported by NIH grant NS42617.
Disclosures
None.
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myocytes. Circ Res. 2009;1047:879–886.
21. Khanna S, Roy S, Maurer M, Ratan RR, Sen CK. Oxygen-sensitive reset of
hypoxia-inducible factor transactivation response: prolyl hydroxylases tune the
biological normoxic set point. Free Radic Biol Med. 2006;4012:2147–2154.
22. Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De Simone
G, et al. Executive summary: heart disease and stroke statistics–2010
update: a report from the American Heart Association. Circulation. 2010;
1217:948–954.
2314 Stroke August 2011
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ONLINE SUPPLEMENT
Supplemental Materials and Methods
Materials
The following materials were obtained from the source indicated: L-glutamic acid
monosodium salt, and lactate dehydrogenase (LDH) cytotoxicity assay kit (SigmaAldrich,
St. Louis, MO); α-tocotrienol (Carotech Inc, Malaysia); DharmaFECTTM
1
transfection reagent (Dharmacon RNA Technologies, Lafayette, CO); Absolutely RNA®
Miniprep kit (Stratagene, La Jolla, CA); mirVanaTM
miRNA isolation kit (Ambion, Austin,
TX); PicoPure RNA Isolation kit (Arcturus, Sunnyvale, CA); dual-luciferase reporter
assay system (Promega Corporation, Madison, WI); calcein acetoxymethyl ester,
LipofectamineTM
LTX and PLUSTM
reagent (Invitrogen Corporation, Carlsbad, CA); 4hydroxynonenal
(HNE) and monoclonal antibody to MRP1 (Enzo Life Sciences,
Plymouth Meeting, PA); rabbit polyclonal MRP1 antibody (Abbiotec, San Diego, CA);
Fluoro-Jade® C (Millipore, Billerica, MA).
For cell culture, Dulbecco’s modified Eagle medium, fetal calf serum, and penicillin and
streptomycin were purchased from Invitrogen Corporation, Carlsbad, CA. Culture dishes
were obtained from Nunc, Denmark.
Cell culture
Mouse hippocampal HT4 neural cells were grown in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 g/ml
streptomycin at 37C in humidified atmosphere of 95% air and 5% CO2 as described
previously1-3
. Glutamate treatment. Immediately before experiments, the culture medium
was replaced with fresh medium supplemented with serum and antibiotics. Glutamate (5
mmol/L for primary neurons and 10 mmol/L for HT4 neural cells) was added to the cell
culture medium as an aqueous solution. No change in the medium pH was observed in
response to the addition of glutamate2,4
. α-Tocotrienol (TCT) treatment. A stock solution
of α-TCT (Carotech Inc, Malaysia) was prepared in ethanol. Before experiments, culture
medium was replaced with fresh medium supplemented with serum and antibiotic, αTCT
was then added to the culture medium as described in the corresponding legends.
Primary cortical neurons
Cells were isolated from the cerebral cortex of rat feti (Sprague-Dawley, day 17 of
gestation; Harlan, Indianapolis, IN) as described previously1,4
. After isolation from the
brain, cells were grown in minimal essential medium supplemented with 10% heatinactivated
fetal bovine serum, 40 μmol/L cystine, and antibiotics (100 μg/ml
streptomycin, 100 units/ml penicillin, and 0.25 μg/ml amphotericin). Cultures were
maintained at 37°C in 5% CO2 and 95% air in a humidified incubator. All experiments
were carried out 24 hours after plating.
siRNA delivery and analysis of genes
Primary cortical neurons (1.5-2.5  106
cells/ well in 12-well plate) or HT4 neural cells
(0.1  106
cells / well in 12-well plate) were seeded in antibiotic free medium for 24h
Natural Vitamin E α-Tocotrienol Protects Against Ischemic Stroke
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prior to transfection. DharmaFECTTM
1 transfection reagent was used to transfect cells
with 100 nmol/L siRNA pool (Dharmacon RNA Technologies, Lafayette, CO) for 72h as
described previously5-7
. For controls, siControl non-targeting siRNA pool (mixture of 4
siRNA, designed to have ≥ 4 mismatches with the corresponding gene) was used. HT4
cells were harvested and re-seeded for treatment with glutamate or α-TCT as indicated
in the respective figure legends. For the primary neurons, media were changed after
24h of seeding. Neurons were treated with glutamate or α-TCT as indicated after 72h of
transfection. For determination of mRNA expression after siRNA transfection, total RNA
was isolated from cells using the Absolutely RNA®
Miniprep kit (Stratagene, La Jolla,
CA). The abundance of mRNA for MRP1 was quantified using real time PCR using
SYBR green-I (Applied Biosystems, Forster City, CA). The following primer sets were
used: m_MRP1_F, 5’-GGT CCT GTT TCC CCC TCT ACT TCT T-3’; m_MRP1_R, 5’GCA
GTG TTG GGC TGA CCA GTA A-3’; m_GAPDH_F, 5’-ATG ACC ACA GTC CAT
GCC ATC ACT-3’; m_GAPDH_R, 5’-TGT TGA AGT CGC AGG AGA CAA CCT-3’;
r_MRP1_F, 5’-TGA ACC ATG AGT GTG CAG AAG GT-3’; r_MRP1_R, 5’-TCA CAC
CAA GCC AGC ATC CTT-3’; r_GAPDH_F, 5’- TAT GAC TCT ACC CAC GGC AAG
TTC A-3’; r_GAPDH_R, 5’- CAG TGG ATG CAG GGA TGA TGT TCT-3’.
miRIDIAN microRNA (miR) mimic/ inhibitor delivery
Primary cortical neurons (1.5-2.5  106
cells/ well in 12-well plate) or HT4 neural cells
(0.1  106
cells / well in 12-well plate) were seeded in antibiotic free medium for 24h
prior to transfection. DharmaFECTTM
1 transfection reagent was used to transfect cells
with miRIDIAN rno-miR-199a-5p mimic / rno-miR-199a-5p inhibitor or mmu-miR-199a-
5p mimic / mmu-miR-199a-5p hairpin inhibitor (Dharmacon RNA Technologies,
Lafayette, CO) as per the manufacturer’s instructions. miRIDIAN miR mimic or inhibitor
negative controls (Dharmacon RNA Technologies, Lafayette, CO) were used for control
transfections. Samples were collected after 72h of miR mimic/inhibitor delivery for
quantification of miR, mRNA and protein expression as described8
.
pGL3-MRP1-3’UTR luciferase reporter assay
miRIDIAN mmu-miR-199a-5p mimic or mmu-miR-199a-5p hairpin inhibitor was
delivered to HT4 neural cells followed by transfection with pGL3-MRP1-3’UTR firefly
luciferase expression construct (Signosis, Sunnyvale, CA) together with renilla
luciferase pRL-cmv expression construct using LipofectamineTM
LTX PLUSTM
reagent.
Luciferase assay were performed using the dual-luciferase reporter assay system
(Promega, Madison, WI). Firefly luciferase activity was normalized to renilla luciferase
expression for each sample as described8
.
Quantification of microRNA expression
Total RNA including miR fraction was isolated using miRVanaTM
miRNA isolation kit
(Ambion, Austin, TX), according to the manufacturer’s protocol. miR-199a-5p levels
were quantified using Taqman Universal Master Mix (Applied Biosystems, Forster City,
CA). miR levels were quantified with the 2(-∆∆CT)
relative quantification method using
miR-16 as the house keeping miR7-10
.
Western blot
Natural Vitamin E α-Tocotrienol Protects Against Ischemic Stroke
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After protein extraction, the protein concentration was determined using BCA protein
reagents. The samples (40-50 μg of protein / lane) were separated on a 4-12% SDSpolyacrylamide
gel electrophoresis as described2,11-12
and probed with anti-MRP1 (1:20
dilution, Enzo Life Sciences, Plymouth Meeting, PA). To evaluate the loading efficiency,
membranes were probed with anti-GAPDH antibody (Sigma-Aldrich, St. Louis, MO).
Cell viability
The viability of primary cortical neurons or HT4 neural cells in culture was assessed by
measuring leakage of lactate dehydrogenase (LDH) from cells into media using an in
vitro toxicology assay kit from Sigma-Aldrich (St. Louis, MO, USA). The protocol has
been described in detail in a previous report5-6,11,13
. In brief, LDH leakage was
determined using the following equation: % total LDH leaked = (LDH activity in the cell
culture medium / total LDH activity )  100. Total LDH activity represents the sum of
LDH activities in the cell monolayer, detached cells, and the cell culture medium.
Immunocytochemistry
HT4 neural cells (0.5  106
cells / well) were seeded in 35mm plates for 24h then
treated with or without 10 mmol/L glutamate. Cells were washed with PBS three times
and fixed in 10% buffered formalin for 20 min, then underwent permeabilization using
0.1% Triton X-100/PBS for 15 min. The cells were washed and incubated with 10% goat
serum (Vector Laboratories, Burlingame, CA) for 1h at room temperature, and
incubated with MRP1 antibody (1:50, Abcam, Cambridge, MA) overnight at 4C. After
incubation with primary antibody, cells were washed with PBS three times and
incubated with an Alexa-flour 488 (1:200 dilution) for 1h at room temperature. After
three washes and incubation with 4’,6’-diamino-2-phenylindole (1:10,000 dilution) for 2
min, cells were mounted in gelmount (aqueous mount, Vector Laboratories, Burlingame,
CA) for microscopic imaging as described previously5,11
.
Calcein clearance assay
Calcein clearance assay was used to measure MRP1 activity in both primary cortical
neurons and HT4 neural cells against glutamate insult. To improve poor specificity of
pharmacological inhibitors, RNA interference approach was applied to measure specific
MRP1 activity. After 72h of MRP1 siRNA transfection as described above, HT4 cells
were re-seeded and treated with glutamate as described in relevant legends. After
glutamate challenge, calcein-AM (25 nmol/L) was loaded to the cells for 15 min at 37°C.
Cells were washed with PBS, collected and analyzed using the Accuri C6TM
(Accuri
Cytometers, Ann Arbor, MI) flowcytometer. MRP1 activity was measured on the basis of
intracellular calcein retention14
. For primary neurons, media were changed after 72h of
transfection, and the neurons were treated with 5 mmol/L glutamate for 1h. After
glutamate challenge, calcein-AM (5 μmol/L) was loaded to the cells for 30 min at 37°C.
Then, fluorescence was measured by using the Synergy 2 Multi-Mode Microplate
Reader (BioTek, Winooski, VT) with the excitation wavelength of 485nm and the
emission wavelength of 528nm.
Mouse stroke model
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Transient focal cerebral ischemia was induced in 8 weeks old MRP1 deficient (n=20,
male, Taconic, Hudson, NY) or background FVB mice (n=15, male, Taconic, Hudson,
NY) by middle cerebral artery occlusion (MCAO) as previously described1,11,15-16
.
Occlusion of the right middle cerebral artery was achieved by using the intraluminal
filament insertion technique. Briefly, mice were anesthetized by inhaling halothan, and
6-0 nylon monofilament was inserted into the internal carotid artery, via the external
carotid artery. Then the filament tip was positioned for occlusion at a distance of 6mm
beyond the internal carotid artery-pterygopalatine artery bifurcation. We observed that
this approach results in a 70 ± 10% drop in cerebral blood flow as measured by laser
Doppler (DRT4, Moor Instruments). Once the filament was secured, the incision was
sutured and the animal was allowed to recover from anesthesia in its home cage. After
90min of occlusion, the animal was briefly re-anesthetized, and reperfusion was initiated
via withdrawal of the filament from MCA. This surgical protocol typically results in a core
infarct limited to the parietal cerebral cortex and caudate putamen of the right
hemisphere. After 48h of reperfusion, T2-weighted image was taken to measure infarct
volume. Mice suffering from surgical complications (e.g. hemorrhage or death) during
MCAO were excluded. Immediately after imaging, tissues from control and strokeaffected
hemispheres of MRP1 (n=12) and FVB (n=8) were harvested.
MRI
T2-weighted imaging was performed on stroke-affected mice. Imaging experiments
were carried out using a 11.7T (500 MHz) MR system comprised of a vertical bore
magnet (Bruker Biospin, Ettlingen, Germany) as described previously by our group11,17-
18
. Briefly, animals were placed inside a 30 mm radio frequency coil (resonator) and
finally the whole arrangement was placed inside the vertical magnet. Shimming was
performed to adjust field inhomogeneities on the subject. After several localizer scans
were completed for three different orientations (saggital, coronal and axial), a T2weighted
spin echo rapid acquisition with relaxation enhancement (RARE) sequence
was optimized for some key parameters such as: field of view (FOV) = 30×30 mm,
acquisition matrix 256×256, repetition time (TR) = 3000 ms, echo time (TE) = 30 ms, flip
angle (FA) = 180 degrees, images in acquisition = 15, resolution = 8.533 pixels/mm, and
number of averages 4 was used to acquire T2-weighted MR images from the mouse
head on the 11.7-T MRI system to generate 15 images corresponding to 15 short axis
slices. For stroke-volume calculations, raw MRI images were converted to digital
imaging and communications in medicine (DICOM) format and read into ImageJ
software (NIH).
α-Tocotrienol supplementation
C57BL/6 (5 weeks, male, Harlan, Indianapolis, IN) mice were randomly divided into two
groups, control (n=18) and supplemented (n=23) group. The control group was orally
gavaged with vitamin E stripped corn oil with volume matching the mean volume of the
supplement in the test group. Stock solution of α-TCT supplement solution was
prepared in vitamin E-stripped corn oil. The test group was orally gavaged with α-TCT
(Carotech Inc, Malaysia) in oil (same as placebo) at a dosage of 50 mg/kg body weight
for 13 weeks. Incorporation of orally supplemented vitamin E to the brain is a slow
process. Longer supplementation period improves bioavailability of vitamin E to the
Natural Vitamin E α-Tocotrienol Protects Against Ischemic Stroke
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brain. Stroke was performed at 20 to 24h after the last supplementation of α-TCT or
corn oil. After 48h of MCAO, T2-weighted image was taken to measure infarct volume.
Mice suffering from surgical complications (e.g. hemorrhage or death) during MCAO
were excluded. Immediately after imaging, tissues from control and stroke-affected
hemispheres of control (n=9) and α-TCT fed (n=10) mice were harvested. Mice were
maintained under standard conditions at 22+2°C with 12:12 dark: light cycles. All animal
protocols were approved by the Institutional Animal Care and Use Committee (IACUC)
of the Ohio State University, Columbus, Ohio.
HPLC-electrochemical detection
Vitamin E analyses of TCT or corn oil fed mice brains were performed using a HPLCcoulometric
electrode array detector (Coularray Detector - model 5600 with 12 channels;
ESA Inc., Chelmsford, MA). This system enables the simultaneous detection of TCTs
and TCPs in the same run as described by us previously2,19
. Glutathione measurement
was performed using an HPLC system coupled with a electrochemical coulometric
detector as described1,4,20
. The CoulArray detector employs multiple channels set at
specific redox potentials. Data were collected using channels set at 600, 700, and
800mV. The samples were snap-frozen and stored in liquid nitrogen until HPLC assay.
Sample preparation, composition of the mobile phase, and specification of the column
used were as described previously2,20
.
mRNA expression assay from laser-captured microdissected somatosensory
cortex of brain tissue
Laser microdissection and pressure catapulting (LMPC) was performed using the
microlaser system from PALM Microlaser Technologies AG (Bernreid, Germany) as
described8,21-23
. Briefly, mice were euthanized immediately after MRI imaging and
coronal slices of brain tissue were collected using a mouse brain matrix. OCTembedded
in slices were subsequently cut in 12μm thick sections on a Leica CM 3050
S cryostat (Leica Microsystems, Wetzlar, Germany). Settings used for laser cutting were
UV-Energy of 70-80 and UV-Focus of 70. Matched area (2  106
μm2
) of contralateral
or stroke-affected somatosensory cortex was captured into 25μl of RNA extraction
buffer. The total RNA was isolated using PicoPure RNA Isolation kit (Arcturus,
Sunnyvale, CA) to measure MRP1 mRNA expression. To determine miR-199a-5p
expression in MCAO-induced mouse brain, miR fraction was isolated using miRVanaTM
miR isolation kit (Ambion, Austin, TX) as described above.
Histology
OCT-embedded frozen brain was sectioned (12μm) and mounted onto slides. brain
sections were stained with rabbit polyclonal antibody to MRP1 (1: 200, Abbiotec, San
Diego, CA), 0.0001% Fluoro-Jade® C (Millipore, Billerica, MA) or 4-HNE (1:1000, Enzo
Life Sciences, Plymouth Meeting, PA). Coronal slices of cortical sections were analyzed
by fluorescence microscopy (Axiovert 200M, Zeiss, Göttingen, Germany) and images
were captured using Axiovert v4.8 software (Zeiss)6,24
.
Statistics
Natural Vitamin E α-Tocotrienol Protects Against Ischemic Stroke
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Data are reported as mean  SD of at least three independent experiments. Difference
in means was tested using Student’s t-test or one-way ANOVA with Tukey’s test.
P<0.05 was considered statistically significant.
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Supplemental Figures and Figure Legends
Figure S1. MRP1 knockdown attenuated the neuroprotection of α-tocotrienol. A,
Glutamate (10mmol/L, 12h) challenge induced the expression of MRP1 in HT4 neural
cells (blue-DAPI stained nuclei; green-MRP1 protein). After MRP1 siRNA transfection,
MRP1 mRNA expression was significantly down-regulated (B). C, Cells were re-split
after transfection. α-TCT (1μmol/L) was added into cell culture medium 6h before
glutamate treatment. After glutamate (10mmol/L, 12h) challenge, LDH leakage was
measured. Neuroprotection of α-TCT was compromised under conditions of MRP1
knockdown. D, Glutamate-challenged cells with MRP1 knockdown exhibited loss of
functional MRP1 by retaining more calcein compared to corresponding control cells.
Bar=50μm. n=3, *
P<0.05 compared with control; §
P<0.05 compared with control siRNAtransfected,
glutamate-treated HT4 cells.
Natural Vitamin E α-Tocotrienol Protects Against Ischemic Stroke
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Figure S2. miR-199a-5p targets MRP1 expression. After 72h of miR-199a-5p mimic
delivery, miR-199a-5p expression was significantly upregulated while miR-199a-5p
hairpin inhibitor significantly downregulated miR-199a-5p in HT4 neural cells (A). miR-
199a-5p mimic/inhibitor negatively regulated mRNA (B), and protein (C) of MRP1. To
test whether MRP1 is a direct target of miR-199a-5p, HT4 cells were transfected with a
pGL3-MRP1-3’UTR firefly luciferase expression construct and co-transfected with
control renilla luciferase reporter construct along with miR-199a-5p mimic or inhibitor.
miR-199a-5p mimic delivered cells showed lower luciferase activity while miR-199a-5p
hairpin inhibitor delivered cells showed higher luciferase activity (D). n=3, *
P<0.05
compared with control.
Natural Vitamin E α-Tocotrienol Protects Against Ischemic Stroke
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Figure S3. Orally supplemented α-tocotrienol did not change brain α-tocopherol
levels or cerebral blood flow. A, C57BL/6 mice were randomly divided into two groups,
and orally gavaged with vitamin E-stripped corn oil (n=18) or 50mg α-TCT per kg body
weight (n=23) for 13 weeks. After 24h of last supplementation, MCAO was performed
for 90min. T2 weighted MRI imaging was performed 48h after reperfusion, then mice
were sacrificed and brain were harvested. B-C, Oral α-TCT supplementation
significantly increased α-TCT level in the brain without affecting brain tissue α-TCP level
(n=5). D, Reduction in the MCA-area blood flow in corn oil fed and α-TCT fed mice
during occlusion was found to be comparable (corn oil, n=9; α-TCT, n=10). *
P<0.05
compared with corresponding control.
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Figure S4. Collection of somatosensory cortical tissue elements using a laser
microdissection and pressure catapulting (LMPC) system. MCAO-challenged
brains were embedded in OCT. OCT-embedded slices were cut in 12μm thick coronal
section. Matched area (2  106
μm2
) of contralateral or stroke-affected cortex was
captured using a LMPC system (A, control hemisphere; B, infarct hemisphere).
Bar=50μm.
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Reference for Supplementary Material
1. Khanna S, Roy S, Slivka A, Craft TK, Chaki S, Rink C, Notestine MA, DeVries AC,
Parinandi NL, Sen CK. Neuroprotective properties of the natural vitamin E alphatocotrienol.
Stroke. 2005;3610:2258-64.
2. Sen CK, Khanna S, Roy S, Packer L. Molecular basis of vitamin E action. Tocotrienol
potently inhibits glutamate-induced pp60(c-Src) kinase activation and death of
HT4 neuronal cells. J Biol Chem. 2000;27517:13049-55.
3. Tirosh O, Sen CK, Roy S, Packer L. Cellular and mitochondrial changes in glutamateinduced
HT4 neuronal cell death. Neuroscience. 2000;973:531-41.
4. Khanna S, Roy S, Ryu H, Bahadduri P, Swaan PW, Ratan RR, Sen CK. Molecular
basis of vitamin E action: tocotrienol modulates 12-lipoxygenase, a key mediator
of glutamate-induced neurodegeneration. J Biol Chem. 2003;27844:43508-15.
5. Khanna S, Roy S, Park HA, Sen CK. Regulation of c-Src activity in glutamateinduced
neurodegeneration. J Biol Chem. 2007;28232:23482-90.
6. Khanna S, Roy S, Parinandi NL, Maurer M, Sen CK. Characterization of the potent
neuroprotective properties of the natural vitamin E alpha-tocotrienol. J
Neurochem. 2006;985:1474-86.
7. Shilo S, Roy S, Khanna S, Sen CK. Evidence for the involvement of miRNA in redox
regulated angiogenic response of human microvascular endothelial cells.
Arterioscler Thromb Vasc Biol. 2008;283:471-7.
8. Roy S, Khanna S, Hussain SR, Biswas S, Azad A, Rink C, Gnyawali S, Shilo S,
Nuovo GJ, Sen CK. MicroRNA expression in response to murine myocardial
infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and
tensin homologue. Cardiovasc Res. 2009;821:21-9.
9. Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY. miR-21-mediated tumor growth. Oncogene.
2007;2619:2799-803.
10. Biswas S, Roy S, Banerjee J, Hussain SR, Khanna S, Meenakshisundaram G,
Kuppusamy P, Friedman A, Sen CK. Hypoxia inducible microRNA 210
attenuates keratinocyte proliferation and impairs closure in a murine model of
ischemic wounds. Proc Natl Acad Sci U S A. 2010;10715:6976-81.
11. Park HA, Khanna S, Rink C, Gnyawali S, Roy S, Sen CK. Glutathione disulfide
induces neural cell death via a 12-lipoxygenase pathway. Cell Death Differ.
2009;168:1167-79.
12. Khanna S, Parinandi NL, Kotha SR, Roy S, Rink C, Bibus D, Sen CK. Nanomolar
vitamin E alpha-tocotrienol inhibits glutamate-induced activation of
phospholipase A2 and causes neuroprotection. J Neurochem. 2010;1125:1249-
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13. Han D, Sen CK, Roy S, Kobayashi MS, Tritschler HJ, Packer L. Protection against
glutamate-induced cytotoxicity in C6 glial cells by thiol antioxidants. Am J Physiol.
1997;2735 Pt 2:R1771-8.
14. van de Ven R, de Jong MC, Reurs AW, Schoonderwoerd AJ, Jansen G, Hooijberg
JH, Scheffer GL, de Gruijl TD, Scheper RJ. Dendritic cells require multidrug
resistance protein 1 (ABCC1) transporter activity for differentiation. J Immunol.
2006;1769:5191-8.
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15. DeVries AC, Joh HD, Bernard O, Hattori K, Hurn PD, Traystman RJ, Alkayed NJ.
Social stress exacerbates stroke outcome by suppressing Bcl-2 expression. Proc
Natl Acad Sci U S A. 2001;9820:11824-8.
16. Li X, Blizzard KK, Zeng Z, DeVries AC, Hurn PD, McCullough LD. Chronic
behavioral testing after focal ischemia in the mouse: functional recovery and the
effects of gender. Exp Neurol. 2004;1871:94-104.
17. Ojha N, Roy S, Radtke J, Simonetti O, Gnyawali S, Zweier JL, Kuppusamy P, Sen
CK. Characterization of the structural and functional changes in the myocardium
following focal ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol.
2008;2946:H2435-43.
18. Gnyawali SC, Roy S, McCoy M, Biswas S, Sen CK. Remodeling of the ischemiareperfused
murine heart: 11.7-T cardiac magnetic resonance imaging of contrastenhanced
infarct patches and transmurality. Antioxid Redox Signal.
2009;118:1829-39.
19. Roy S, Venojarvi M, Khanna S, Sen CK. Simultaneous detection of tocopherols and
tocotrienols in biological samples using HPLC-coulometric electrode array.
Methods Enzymol. 2002;352:326-32.
20. Sen CK, Khanna S, Babior BM, Hunt TK, Ellison EC, Roy S. Oxidant-induced
vascular endothelial growth factor expression in human keratinocytes and
cutaneous wound healing. J Biol Chem. 2002;27736:33284-90.
21. Roy S, Khanna S, Azad A, Schnitt R, He G, Weigert C, Ichijo H, Sen CK. Fra-2
mediates oxygen-sensitive induction of transforming growth factor beta in cardiac
fibroblasts. Cardiovasc Res. 2010;874:647-55.
22. Kuhn DE, Roy S, Radtke J, Gupta S, Sen CK. Laser microdissection and pressurecatapulting
technique to study gene expression in the reoxygenated myocardium.
Am J Physiol Heart Circ Physiol. 2006;2906:H2625-32.
23. Kuhn DE, Roy S, Radtke J, Khanna S, Sen CK. Laser microdissection and capture
of pure cardiomyocytes and fibroblasts from infarcted heart regions: perceived
hyperoxia induces p21 in peri-infarct myocytes. Am J Physiol Heart Circ Physiol.
2007;2923:H1245-53.
24. Rink C, Roy S, Khan M, Ananth P, Kuppusamy P, Sen CK, Khanna S. Oxygensensitive
outcomes and gene expression in acute ischemic stroke. J Cereb Blood
Flow Metab. 2010;307:1275-87.