Original Contribution
Resveratrol Preconditioning Protects Against Cerebral
Ischemic Injury via Nuclear Erythroid 2–Related Factor 2
Srinivasan V. Narayanan, BS; Kunjan R. Dave, PhD; Isa Saul, BS; Miguel A. Perez-Pinzon, PhD
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Background and Purpose—Nuclear erythroid 2 related factor 2 (Nrf2) is an astrocyte-enriched transcription factor that
has previously been shown to upregulate cellular antioxidant systems in response to ischemia. Although resveratrol
preconditioning (RPC) has emerged as a potential neuroprotective therapy, the involvement of Nrf2 in RPC-induced
neuroprotection and mitochondrial reactive oxygen species production after cerebral ischemia remains unclear. The goal
of our study was to study the contribution of Nrf2 to RPC and its effects on mitochondrial function.
Methods—We used rodent astrocyte cultures and an in vivo stroke model with RPC. An Nrf2 DNA binding ELISA and
protein analysis via Western blotting of downstream Nrf2 targets were performed to determine RPC-induced activation
of Nrf2 in rat and mouse astrocytes. After RPC, mitochondrial function was determined by measuring reactive oxygen
species production and mitochondrial respiration in both wild-type and Nrf2−/− mice. Infarct volume was measured to
determine neuroprotection, whereas protein levels were measured by immunoblotting.
Results—We report that Nrf2 is activated by RPC in rodent astrocyte cultures, and that loss of Nrf2 reduced RPC-mediated
neuroprotection in a mouse model of focal cerebral ischemia. In addition, we observed that wild-type and Nrf2−/− cortical
mitochondria exhibited increased uncoupling and reactive oxygen species production after RPC treatments. Finally,
Nrf2−/− astrocytes exhibited decreased mitochondrial antioxidant expression and were unable to upregulate cellular
antioxidants after RPC treatment.
Conclusions—Nrf2 contributes to RPC-induced neuroprotection through maintaining mitochondrial coupling and
antioxidant protein expression. (Stroke. 2015;46:00-00. DOI: 10.1161/STROKEAHA.115.008921.)
Key Words: ischemic preconditioning ◼ mitochondria ◼ reactive oxygen species ◼ resveratrol
I
n the United States, 1 in 20 deaths can be attributed to
stroke, whereas >80% of the 800 000 people who experience
stroke each year survive and require long-term rehabilitation.1
Ischemic preconditioning has emerged as a potential therapy
that could mitigate the morbidity of cerebral ischemic injury;
previous studies from our group have shown that ischemic
preconditioning treatment induced neuroprotection in rodent
models of global cerebral ischemia.2,3 This protection has been
recapitulated using the polyphenolic compound resveratrol as a
pharmacological preconditioning agent.4 The use of resveratrol
as a preconditioning agent has been substantiated by numerous
studies in a diverse range of in vitro and in vivo models.5,6
However, previous preconditioning studies have focused
mainly on neuronal physiology and amelioration of neuronal cell death after cerebral ischemia. As a result, the role
of astrocytes in mediating ischemic preconditioning is often
neglected, despite the well-known functions of astrocytes in
mediating several neuroprotective mechanisms.7 Astrocytes
have been suggested to have increased resistance to ischemic
injury when compared with neurons8; however, astrocyte dysfunction has been shown to exacerbate various neurodegenerative conditions9–11 and increase susceptibility of neurons
to ischemia.12 Rodents have both a lower astrocyte:neuron
ratio and fewer astrocytic processes compared with humans.13
Indeed, the relative differences in cytoarchitecture between
rodents and humans may have contributed to the relative plateau of clinically translatable neuroprotective agents.
One of the many neuroprotective functions of astrocytes
includes supplying neurons with antioxidants, the production of which is primarily controlled by the transcription factor nuclear erythroid 2 related factor 2 (Nrf2). Nrf2 has been
previously suggested to be highly expressed in astrocytes as
opposed to neurons and has been shown to increase the antioxidant proteins thioredoxin and NAD(P)H-quinone oxidoreductase 1 (NQO-1).14 Because oxidative stress is a major
consequence of cerebral ischemia, the function of Nrf2 to mitigate this stress makes Nrf2 and related downstream pathways
attractive targets to combat cerebral ischemic injury.
Received January 24, 2015; final revision received February 26, 2015; accepted March 17, 2015.
From the Cerebral Vascular Disease Research Laboratories (S.V.N., K.R.D., I.S., M.A.P.-P.), Neuroscience Program (S.V.N., K.R.D., M.A.P.-P.), and
Department of Neurology (S.V.N., K.R.D., I.S., M.A.P.-P.), University of Miami Miller School of Medicine, FL; and University of Miami Miller School
of Medicine MD/PhD Program (S.V.N.).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.
115.008921/-/DC1.
Correspondence to Miguel A. Perez-Pinzon, PhD, Department of Neurology, D4-5, University of Miami Miller School of Medicine, PO Box 016960,
Miami, FL 33101. E-mail perezpinzon@miami.edu
© 2015 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.115.008921
1
2 Stroke June 2015
In light of the aforementioned studies, the focus of our investigation was to determine whether resveratrol preconditioning
(RPC) treatment could induce neuroprotection through Nrf2
activation. We found that the absence of functional Nrf2 protein reduced RPC-induced neuroprotection in a mouse model
of focal cerebral ischemia. In addition, RPC treatment failed
to increase mitochondrial and cellular antioxidants in cultured
astrocytes when Nrf2 was absent. These studies highlight the
contribution of astrocyte Nrf2 to RPC-induced protection and
a novel role of Nrf2 in maintaining mitochondrial function.
Methods
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Additional detailed methods are described in the online-only Data
Supplement. All animal protocols were approved by the Animal Care
and Use Committee of the University of Miami. Minimum Essential
Medium, Hanks’ Balanced Salt Solution, and fetal bovine serum were
purchased from Life Technologies (Grand Island, NY). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless
otherwise noted.
Preparation of Primary Cultures and In Vitro
Preconditioning Studies
Astrocyte cultures were prepared as previously described.15 Cortical
tissue was harvested from postnatal day 2–4 Sprague Dawley rats,
C57Bl/6J wild-type (WT) mice, or Nrf2−/− mice. After treatment with
0.25% trypsin and 0.1% DNase, single cell suspensions were plated
onto cell-culture dishes and maintained for 10 to 14 days before experimental use. After reaching ≈80% confluence, cultures were trypsinized and passaged. Passages 1 to 3 were used for experiments. For
RPC treatment, astrocyte cultures were exposed to 2 hours of resveratrol (25 μmol/L) or dimethyl sulfoxide (vehicle) 48 hours before
harvesting cell lysates or nuclear fractions for downstream analysis.
ELISA and Western Blotting
Nuclear and cytoplasmic fractionations were prepared according to the
manufacturer’s protocol using a nuclear extract kit (Active Motif Cat.
No.: 40010). Nuclear and cytoplasmic extracts were probed with both
Lamin B and GAPDH to establish purity of the nuclear and cytoplasmic
fractions, respectively. A total of 10 μg of nuclear samples were used for
the TransAM Nrf2 ELISA kit (Active Motif) to measure DNA binding
of activated Nrf2 nuclear protein, as determined by absorbance measurements at 450 nm. For immunoblotting, cells were lysed in radioimmunoprecipitation assay buffer and immunoblotted for Nrf2, uncoupler
protein 2 (UCP2), GAPDH, β-actin, manganese superoxide dismutase
(MnSOD), or Lamin B. Proteins were detected using enhanced chemiluminescence system (Pierce, ThermoScientific) and densitometry was
performed using ImageJ (National Institute of Health).16
Animal Model of Focal Cerebral Ischemia
For our focal cerebral ischemia model, the left middle cerebral artery
(MCA) was occluded for 1 hour using an intraluminal filament model
as previously described.17 C57Bl/6J WT or Nrf2−/− male mice between
7 and 11 weeks were randomly assigned to 2 treatment groups: resveratrol (10 mg/kg IP) or dimethyl sulfoxide (vehicle). The investigator
was blinded to the administration of these agents 48 hours before MCA
occlusion (MCAO). Twenty-four hours after reperfusion, infarct volume was assessed with 2,3,5-triphenyltetrazolium chloride (TTC) and
quantified using ImageJ software. Exclusion criteria for these studies
included (1) >30% of baseline MCA blood flow persisting through the
occlusion phase of the MCAO injury as measured by laser Doppler
flowmetry and (2) lack of detectable infarct after TTC staining.
Polarography
Mitochondrial respiration studies were conducted as previously described.18 In brief, nonsynaptic mitochondria were isolated from WT
or Nrf2−/− mice 48 hours after resveratrol or vehicle treatment. The
ratio of state III/state IV respiration was measured using a Clark-type
oxygen electrode. This ratio represented the respiratory control index
(RCI), an established measure of mitochondrial coupling.19
Measurement of Mitochondria ROS Production
Mitochondrial ROS production was determined using a spectrophotometer following a previously established protocol.20 Isolated nonsynaptic mitochondria from Nrf2−/− or WT animals were added to a
microplate containing horseradish peroxidase, Amplex Red Ultra,
and superoxide dismutase. H2O2 emission was measured spectrophotometrically at 555 nm excitation/590 nm emission wavelengths.
After establishing baseline measurements, respiratory substrates were
added in a similar manner to the polarographic studies. Rates of H2O2
emission were recorded for each complex-specific substrate/inhibitor
pair and normalized to baseline H2O2 production for each sample.
Statistical Analysis
All data are expressed as mean±SD. Statistical analysis between 2
groups was performed using the unpaired Student t test. Statistical
analysis between >2 groups was performed using a 1-way ANOVA
with Bonferroni multiple comparison post hoc test unless otherwise
specified. P≤0.05 was considered statistically significant (GraphPad
Prism v5.00 for Windows, GraphPad Software, San Diego, CA).
Results
Loss of Nrf2 Decreases RPC-Induced
Neuroprotection After Focal Cerebral Ischemia
Previous studies from our laboratory have shown that RPC
can induce neuroprotection against cerebral ischemia in
vivo.4 Therefore, we wanted to determine whether the loss
of functional Nrf2 decreased RPC-induced neuroprotection
in a mouse model of focal cerebral ischemia. Nrf2−/− mice
were verified by standard genotyping and immunoblotting for
NQO-1 (Figure I in the online-only Data Supplement). WT
and Nrf2−/− mice were subjected to MCAO injury 48 hours
after either RPC or vehicle treatment. Quantification of TTCstained brain slices from each treatment group indicated that
RPC treatment of WT mice significantly reduced infarct volume compared with vehicle treatment (44.43±13.90%, n=9
versus 28.31±9.93%, n=6, respectively; P<0.05; Figure 1A
and 1B). However, no significant difference was observed
in Nrf2−/− mice between RPC and vehicle-treated groups
(40.86±17.10%, n=10 versus 34.47±14.25%, n=9; P=0.39).
In addition, there was no significant difference between WT
and Nrf2−/− vehicle-treated groups. Therefore, the results
from Figure 1 indicate that RPC-induced neuroprotection was
decreased in the absence of functional Nrf2.
Loss of RPC-Induced Neuroprotection in Nrf2−/−
Mice Is Not Due to Altered Cerebral Blood Flow
A previous group has suggested that the cerebral blow flow is
altered in Nrf2−/− mice, which led to their use of a modified in
vivo model to achieve adequate MCA occlusion.21 To determine whether the observed infarct volumes from Figure 1
were attributable to strain differences in cerebral blood flow,
we analyzed blood flow throughout the MCAO injury via
laser Doppler flowmetry for each treatment group of mice.
The average blood flow for each group during the phases
of the MCAO injury (baseline, occlusion, and reperfusion)
Narayanan et al Effect of Nrf2 on Resveratrol Preconditioning 3
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Figure 1. Resveratrol preconditioning (RPC)–induced neuroprotection is decreased in nuclear erythroid 2–related factor 2
(Nrf2)−/− mice after middle cerebral artery occlusion (MCAO) injury.
A, Representative image of triphenyltetrazolium chloride staining of brain slices to assess infarct volume of mice subjected to
MCAO injury. Slices are distributed rostrocaudally. B, Quantification of infarct volume. Areas of cortical infarction at 7 coronal
levels were cumulated and normalized for edema. Number of
subjects represented in inset of bar graph. Data are presented as
mean±SD; *P≤0.05, (multiple comparison 1-way ANOVA followed
by Bonferroni test). VEH indicates vehicle; and WT, wild type.
were compared and expressed as a percentage of baseline
laser Doppler Flowmetry measurements (Figure 2A); our
results show that there was no statistically significant difference between blood flow of any of the treatment groups for
each phase of the MCAO injury. In Figure 2B, the average
age and weight for each treatment group were analyzed and
once again there was no statistically significant differences
observed. Therefore, the loss of RPC-induced neuroprotection in Nrf2−/− mice is not attributable to altered cerebral
blood flow.
RPC Treatments Activate Nrf2 in Rat Astrocytes
Given that the reduction in RPC-induced neuroprotection
in Nrf2−/− mice was not due to altered cerebral blood flow
(Figure 2), the results from Figure 1 suggest that RPCinduced neuroprotection partially requires Nrf2. As there is
greater Nrf2 protein abundance in astrocytes,14 we next sought
to determine whether RPC can activate Nrf2 in vitro using
astrocyte cultures. Using the TransAM Nrf2 ELISA kit, RPC
treatment increased the amount of activated Nrf2 in nuclear
astrocyte fractions at 48 hours compared with vehicle-treated
cultures as determined by absorbance measured at 450 nm
Figure 2. Reduced resveratrol preconditioning (RPC)–induced
neuroprotection in nuclear erythroid 2–related factor 2 (Nrf2)−/−
mice is not because of altered cerebral blood flow during ischemia. A, Blood flow measurements during the baseline, ischemic/
occlusion, and reperfusion phases of the middle cerebral artery
occlusion (MCAO) injury from the same treatment subjects as
in Figure 1. Data represented as a percentage of baseline blood
flow, as measured by laser Doppler flowmetry. B, Physiological
variables of WT and Nrf2−/− mice used for MCAO experiments.
WT indicates wild type.
(0.983±0.458 versus 0.625±0.352; P<0.05; n=3; Figure 3A),
which were not observed at earlier time points (1 or 24 hours)
after RPC treatment. To determine whether downstream pathways of Nrf2 were increased after RPC, whole-cell astrocyte
lysates were probed for NQO-1, an Nrf2-dependent gene
target. 48 hours after RPC treatments, NQO-1 protein levels
were significantly increased in astrocyte cultures by ≈2.1 fold
compared with vehicle treatments (Figure 3B). The results
indicate that RPC activates astrocyte Nrf2 by increasing Nrf2
DNA binding and Nrf2-dependent gene transcription.
RPC Induces Uncoupling in WT and Nrf2−/−
Mitochondria
Resveratrol has previously been implicated in modifying
cerebral mitochondrial function. Therefore, we investigated
mitochondrial coupling (represented as RCI) in isolated nonsynaptic mitochondria from WT and Nrf2−/− mouse cortex
after RPC treatment in vivo. We used nonsynaptic mitochondria because this fraction contains more astrocyte-derived
mitochondria than the synaptic fraction and therefore would
better represent functional changes because of the loss of
Nrf2. RPC-induced treatment induced a mild uncoupling in
both WT and Nrf2−/− mitochondria compared with vehicle
treatments of each respective mouse strain (WT: vehicle
5.84±0.55 versus RPC 3.93±0.42; P<0.05 and Nrf2−/−: vehicle
3.272±0.67 versus RPC 2.27±0.15; P<0.05; Figure 4A). In
addition, we observed a significant difference between Nrf2−/−
and WT mice RCI values between vehicle groups, suggesting
that Nrf2−/− mitochondria have an innate respiratory dysfunction. Therefore, RPC treatment had similar effects on the RCI
in both mouse strains, with RPC treatment further reducing
the already decreased RCI in Nrf2−/− mice.
4 Stroke June 2015
Figure 3. Reduced resveratrol preconditioning (RPC) treatment activates nuclear erythroid 2–related factor 2 (Nrf2) in astrocyte cultures.
A, ELISA assay was used to measure Nrf2 DNA binding in rat astrocyte nuclear fractions harvested at 1, 24, or 48 hours after RPC treatment. B, whole-cell astrocyte lysates were probed for NAD(P)H-quinone oxidoreductase 1 (NQO-1) expression after RPC treatment. n=4,
*P≤0.05 and **P≤0.01.
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RPC Induces Increased UCP2 Expression
in Nrf2−/− Astrocytes
RPC Treatment Increases ROS Production in WT
and Nrf2−/− Mitochondria
Previous studies from our laboratory have suggested an
interaction between resveratrol and UCP2, an uncoupler
protein which if altered by RPC could explain RCI values and provide a molecular understanding for the results
observed in Figure 4A. As isolation of astrocyte mitochondria from the brain was not feasible, we took advantage of
highly enriched astrocyte cultures to look at the effects of
Nrf2 on UCP2 expression in vitro. Therefore, 48 hours after
RPC treatment, whole cell mouse WT and Nrf2−/− astrocyte
lysates were prepared and probed for UCP2. Compared
with vehicle treatments, UCP2 was significantly increased
48 hours after RPC treatment in WT and Nrf2−/− astrocyte
cultures (4.05±1.01-fold and 1.89±0.26-fold, respectively;
P<0.05; Figure 4B and 4C). Therefore, RPC treatment
increased UCP2 protein expression in WT and Nrf2−/−
mouse astrocyte cultures.
As Nrf2−/− mice are expected to have decreased antioxidant
capacity, we also measured mitochondrial H2O2 production as
a measure of ROS generation in nonsynaptic mitochondria isolated from WT and Nrf2−/− mouse cortex after RPC treatment in
vivo. RPC treatment significantly increased the fold of H2O2 production compared with baseline production following rotenoneinduced complex I inhibition (WT: vehicle 3.66±1.19 versus RPC
8.29±1.56-fold of baseline P<0.05 and Nrf2−/−: vehicle 3.90±2.82
versus RPC 8.35±2.65-fold; P<0.05) and antimycin-induced
complex III inhibition (WT: vehicle 7.54±2.43 versus RPC
31.06±10.17; P<0.005 and Nrf2−/−: vehicle 9.59±6.32 versus
RPC 23.25±10.05; P<0.05) for both strains of mice (Figure 5A).
However, there were no significant differences between WT and
Nrf2−/− mice for the same treatment group. These findings suggest a role of RPC in increasing ROS production at complex I and
III in WT and Nrf2−/− cortical mitochondria.
Figure 4. Resveratrol preconditioning (RPC) treatment increases mitochondrial uncoupling in nuclear
erythroid 2–related factor 2 (Nrf2)−/− and wild-type
(WT) mice. A, Respiratory control index (RCI) of WT
and Nrf2−/− nonsynaptic mitochondria after RPC
and vehicle treatment. n=4, *P≤0.05 (WT vehicle vs
WT RPC); #P≤0.05 (WT vehicle vs Nrf2−/− vehicle);
^
P≤0.05 (Nrf2−/− vehicle vs Nrf2−/− RPC. B, Uncoupler protein 2 (UCP2) and actin protein levels from
WT and Nrf2−/− astrocyte cultures treated with RPC
and vehicle. (+) positive control: mouse cortex tissue lysate. C, Quantification of UCP2 protein levels
normalized to actin. n=3, *P≤0.05.
Narayanan et al Effect of Nrf2 on Resveratrol Preconditioning 5
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Figure 5. Resveratrol preconditioning (RPC) increases nonsynaptic mitochondrial reactive oxygen species production and only increases
antioxidant expression in wild-type (WT) astrocytes. A, Complex I and complex III H2O2 production rate in WT or nuclear erythroid
2–related factor 2 (Nrf2)−/− nonsynaptic mitochondria. n=4 to 6, *P≤0.05 (WT vehicle vs WT RPC); #P≤0.05 (Nrf2 vehicle vs Nrf2−/− RPC).
B, Western blots of astrocyte cultures for manganese superoxide dismutase (MnSOD), NAD(P)H-quinone oxidoreductase 1 (NQO-1), and
actin (loading control) 48 hours after RPC or vehicle treatment. C, Quantification of WT astrocyte NQO-1 levels and (D) WT and Nrf2−/−
astrocyte MnSOD protein levels, normalized to actin protein levels. n=3 to 6. *P<0.05 and **P<0.005.
RPC-Induced Antioxidant Enzyme Expression in
WT and Nrf2−/− Astrocytes
Although RPC-induced increase in mitochondrial ROS production occurred in both WT and Nrf2−/− mice, we hypothesized that this phenomenon was detrimental in Nrf2−/− mice and
could explain loss of RPC-induced neuroprotection in this population. Therefore, we immunoblotted WT and Nrf2−/− astrocyte culture lysates for the antioxidants MnSOD and NQO-1
48 hours after RPC treatment. (Figure 5B). RPC treatment
significantly increased NQO-1 protein expression (normalized to actin) in WT astrocytes compared with vehicle treatment (1.91±0.35-fold increase; n=6; P<0.05). As expected, we
were unable to observe or measure the Nrf2-dependent protein
NQO-1 in Nrf2−/− astrocyte lysates (Figure 5B and 5C). Next,
we measured the mitochondrial antioxidant MnSOD proteins
levels in WT and Nrf2−/− astrocyte cultures via Western blotting.
We found no statistically significant difference in MnSOD protein levels between vehicle and RPC-treated groups of either
strain of mice (Figure 5D). However, vehicle and RPC-treated
Nrf2−/− astrocytes had significantly less MnSOD protein compared with WT vehicle MnSOD levels (40.3% and 37.2% less
MnSOD protein, respectively; P≤0.005). These results suggest that Nrf2−/− astrocytes have depressed mitochondrial and
cellular antioxidants and are unable to upregulate these proteins in response to RPC-induced ROS production observed
in Figure 5A.
Discussion
In the present study, we investigated the contribution of Nrf2
to RPC-induced neuroprotection and the effect of Nrf2 on
6 Stroke June 2015
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cortical mitochondrial function. We investigated the role of
Nrf2 in RPC-induced neuroprotection in a rodent model of
focal cerebral ischemia. Our MCAO studies suggest that in
the absence of Nrf2 protein, RPC-induced neuroprotection
is not effective in significantly ameliorating cerebral infarction in mice (Figure 1). Given that RPC treatment increased
ROS production in both WT and Nrf2−/− cortical mitochondria
(Figure 4B), and that RPC was unable to induce antioxidant
expression in Nrf2−/− mice (Figure 5), we believe this evidence
suggests that RPC-induced neuroprotection can be attributed
to ROS-mediated signaling pathways, which ultimately activates astrocytic Nrf2 and confers cerebral ischemic tolerance.
The role of astrocyte pathways has not been fully elucidated
in preconditioning research, but their importance to neuronal
disease has been extensively studied. Previous studies by Bell
et al22 highlighted that astrocytic Nrf2 was necessary for ischemic tolerance in murine neuronal cultures. In direct contrast,
Haskew-Layton et al23 determined that physiological levels of
H2O2 could induce ischemic tolerance in neurons independent
of Nrf2. Although our study is a different preconditioning
paradigm (ie, RPC), our current studies indicate that RPC is
indeed able to increase Nrf2 DNA binding and downstream
expression of NQO-1, an Nrf2-dependent gene.
Similar to a previous study,21 we did not observe any difference in infarct volume between vehicle-treated WT and
Nrf2−/− mice at 24 hours. In contrast, we observed significant neuroprotection with RPC treatment in WT mice after
MCAO, but there was no significant reduction of infarct volume in RPC-treated Nrf2−/− mice. Although resveratrol has
been shown to activate a multitude of pathways, resveratrol
was still unable to significantly ameliorate infarct injury in
Nrf2−/− mice after MCAO. This suggests that Nrf2 activation
is a key pathway for RPC-induced neuroprotection.
Our current investigation has also shown that resveratrol
induced an increase in ROS production in WT and Nrf2−/−
mitochondria, which can be detected 48 hours after treatment.
This increase in ROS after resveratrol treatment has been seen
previously in yeast cells,24 human adipocytes,24 and cancer cell
lines.25 In addition, the increase in ROS after preconditioning
treatments is a well-observed phenomenon, and inhibition of
ROS production has been shown to ameliorate preconditioning-induced neuroprotective effects.26
We also present findings that RPC treatment increased
UCP2 expression in astrocyte cultures, and that this increase
was several fold more in WT versus Nrf2−/− astrocytes
(Figure 4). Although our previous findings described an RPCinduced decrease in UCP2 protein expression in adult rat
hippocampal mitochondria, our current studies investigated
UCP2 in cortical postnatal mouse astrocyte cultures. This
discrepancy could be because of the use of different models
and cell types, and future studies may serve to elucidate the
dependence of cell type and brain region on the regulation
of UCP2 by RPC treatment. Taken together, we propose that
ROS production from RPC treatment induces UPC2 expression, leading to mild uncoupling and subsequent protection
against oxidative stress. Furthermore, RPC-induced increase
in UCP2 (Figure 4), basal MnSOD, and NQO-1 (Figure 5)
protein levels were all decreased several fold in Nrf2−/− mice
compared with WT mice; we believe that this implicates Nrf2
as a critical pathway in which RPC-mediated mitochondrial
ROS production activates Nrf2, thus promoting the induction of antioxidant pathways and subsequent neuroprotection
against focal cerebral ischemia.
After RPC treatment, WT and Nrf2−/− exhibited reductions
in their RCI, suggestive of a resveratrol-induced uncoupling
effect. By further decreasing the coupling of Nrf2−/− mitochondria, resveratrol may exacerbate already dysfunctional
cortical mitochondria in Nrf2−/− mice. Thus, decreased mitochondrial coupling, increased ROS production, and decreased
antioxidant defenses could be plausible explanations as to
explain why RPC was less effective in Nrf2−/− mice when
compared to WT mice. Our findings that Nrf2−/− nonsynaptic
mitochondria exhibited decreased coupling were in line with
previous studies, which suggested that Nrf2−/− brain, heart,
and liver mitochondria exhibit decreased RCI.27 Interestingly,
studies by Fiskum et al28 did not describe any changes to brain
nonsynaptic mitochondrial respiration after activation of Nrf2
with sulforaphane. Future studies may serve to understand
the relationship of Nrf2 to cortical nonsynaptic and synaptic
mitochondria.
In conclusion, our investigation provides new insight into
the mechanism of RPC-induced protection and implicates
Nrf2 as an important pathway to induce RPC’s neuroprotective effects in the context of stroke.
Sources of Funding
This work was supported by grants from the National Institutes
of Health, National Institute of Neurological Disease and Stroke
NS45676, NS054147, NS34773, F31NS080344, and the Lois Pope
Life Science Research Award. Dr Perez-Pinzon was supported in part
by the University of Miami Miller School of Medicine.
Disclosures
None.
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Resveratrol Preconditioning Protects Against Cerebral Ischemic Injury via Nuclear
Erythroid 2−Related Factor 2
Srinivasan V. Narayanan, Kunjan R. Dave, Isa Saul and Miguel A. Perez-Pinzon
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SUPPLEMENTAL MATERIAL
Materials
Minimum Essential Medium (MEM), Hanks Balanced Salt Solution (HBSS) and Fetal Bovine
Serum (FBS) were purchased from Gibco/Life Technologies (Grand Island, NY). All other
reagents were purchased from Sigma (St. Louis, MO) unless otherwise noted.
Animal Use
All animal protocols were approved by the Animal Care and Use Committee of the University of
Miami. Experiments were conducted in accordance to ARRIVE guidelines. 16-17 day-pregnant
Sprague-Dawley rats were purchased from Charles Rivers Laboratories and housed in a
temperature controlled environment with 12 hr light -12 hr dark cycle and ad libitum food and
water. 5 week old male and female homozygous knockout mice (Nrf2-/-, Jackson Laboratories)
were bred to establish homozygous Nrf2-/- colonies. The targeting vector replaces exon 5 and
part of exon 4 of Nrf2 with a lacZ reporter followed by a neomycin resistant cassette with a
polyadenylation sequence1. This targeted sequence essentially negates the ability of Nrf2 to bind
to its response element, rendering Nrf2 ineffective in upregulating antioxidant gene transcription.
This construct was electroporated into 129X1/SvJ-derived JM-1 embryonic stem (ES) cells.
Chimeric mice with the correct ES-targeted cells were bred with C57BL/6J mice to generate
Nrf2-/- mice. This subsequent strain was backcrossed with C57BL/6J mice for at least 10
generations. DNA was harvested from ear punches of each mouse and analyzed by polymerase
chain reaction (PCR) to confirm its genotype and confirmed by a third-party genotyping service
(Transnetx).
Polymerase Chain Reaction
Standard PCR was used to differentiate Nrf2-/- mice from WT mice according to Jackson
Laboratory protocol. DNA from 2-3 mm ear punch samples were isolated by digestion in 75 µL
lysis buffer (50 mmol/L KCl, 10 mmol/L Tris-HCl pH 9.0, 0.1% Triton X-100, 0.4 mg/ml
Proteinase K). Samples were incubated in lysis buffer for 1 hr at 95°C. After replacing samples
on ice (4°C), 75 µL of neutralization reagent (25 mmol/L 10 N NaOH, 0.2 mmol/L EDTA) was
added and samples were centrifuged for 30 s on a table-top centrifuge at 12000 RPM. For PCR
reactions, 20 µM of each of the two primers (WT reverse: 5’-GGA ATG GAA AAT AGC TCC
TGC C-3’; Nrf2-/- reverse: 5’-GAC AGT ATC GGC CTC AGG AA-3’) were added to
approximately 10 µg of DNA isolated from WT or Nrf2-/- mouse ear punch samples, along with
25 µL of 2x MangoMix (Bioline Inc., USA) and 3.5 mmol/L MgCl2. Samples were
thermocycled according to Jackson Laboratory protocol for Nrf2-/- mouse strain (Stock#017009)
and according to Chan et. al1.
Preparation of embryonic neuronal and post-natal astrocyte cultures
Astrocyte and neuronal cultures were prepared as previously described2 with slight
modifications. For astrocyte cultures. Cortices from postnatal Sprague Dawley rat pups (P2-P4)
were harvested, followed by digestion with 0.25% trypsin and DNase. Following tituration and
filtration through a 70 µm filter, the resulting cell suspension was centrifuged at 2001 g for 5
minutes and plated in Minimal Essentials Media (MEM) supplemented with 20 mmol/L glucose,
1% GlutaMAX, 1% Penicillin/Streptomycin, and 10% FBS before plating. Complete media
changes were performed every 2-3 days until cultures reached 70% confluency 7 days following
the initial plating. Astrocyte cultures were then passaged and plated in appropriate culture vessels
at a density of 50,000 cells/cm2, and allowed to reach full confluency and maintained for an
additional 6-7 days before experimental use.
Neuronal cultures were prepared from embryonic (E15-16) rat pups. Embryonic cortices were
harvested similarly to the post-natal rat pup astrocyte preparation, except titurated cells were
initially plated onto poly-d-lysine-coated 10 cm dishes (2-3 hemispheres/dish) in MEM
supplemented with 20 mmol/L glucose, 1% GlutaMAX, and 5% FBS. 3 days after the initial
plating, cultures were treated with 5 µmol/L cytosine arabinoside for 48 hrs to terminate
proliferation of contaminating cell populations and subsequent half media changes were
performed every 3-4 days using neuronal maintenance media (MEM, 20 mmol/L glucose, 1%
GlutaMAX). Cultures were used at 10-14 days in vitro (DIV) for immunoblotting experiments.
Subcellular fractionation
Subcellular fractionation was performed as previously described3 with minor modifications. For
mitochondrial isolation, cells were suspended in an isotonic buffer consisting of 250 mM
sucrose, 1 mM EDTA, 0.25 mM DTT, and 1 mg/mL Bovine Serum Albumin (BSA, fraction V).
BSA was added to bind free fatty acids, and to improve the degree of coupling of isolated
mitochondria4. Cells were then homogenized using a teflon-glass homogenizer. The resulting
homogenate was centrifuged at 1,000 x g for 5 min at 4°C. The resulting supernatant was further
centrifuged at 13,000 x g for 10 min at 4°C to pellet mitochondria. The supernatant was
collected, and this represented the cytoplasmic fraction of the cells. Mitochondrial pellets were
washed twice with isolation buffer and resuspended in RIPA lysis buffer for Western blot
analysis. Alternatively, mitochondria were resuspended in isolation buffer for mitochondrial
subfractionation, and finally resuspended in 0.25 M sucrose for respiration studies. The nuclear
fraction was isolated using a previously described protocol. In brief, cells plated on 100 mm cellculture treated dishes were washed with 1x PBS and harvested in 1x PBS with protease and
phosphatase
inhibitor cocktail (Sigma). The cells were then centrifuged at 1000 g x 5 min at 4 deg C. The
supernatant was discarded and the remaining cells were resuspended in hypotonic buffer
consisting of 10 mM HEPES, 1.5 mmol/L MgCl210 mM KCl, and 0.5 mmol/L DTT, pH 7.9.
The cells were allowed to swell for 15 minutes, after which NP-40 detergent was added to a final
concentration of 0.1 %v/v and the cell suspension was vortexed vigorously for 10 seconds. The
cell suspension was then centrifuged for 14000 g x 1 minute at 4 deg C, and the supernatant was
collected and also corresponded to the cytoplasmic fraction. The nuclear pellet was washed once
with hypotonic buffer and lysed in lysis buffer (Active Motif cat.# 16965838).
Oxygen Glucose Deprivation
To mimic IPC in vitro, astrocyte cultures were exposed to oxygen and glucose deprivation
(OGD) as previously described5 for 1 hr. Through empirical testing, 1 hr was determined to be a
sublethal duration of OGD that induced the highest degree of protection to astrocytes following a
lethal OGD insult (6 hrs). The 6 hr time point was chosen because greater than 50% cell death
occurred along with minimal cell detachment from the tissue culture dishes, allowing for more
accurate lactate dehydrogenase release assays. To induce OGD, cells were washed two times
with glucose-free HBSS (in mmol/L: CaCl2 1.26, KCl 5.37, KH2PO4 0.44, MgCl2 0.49, MgSO4
0.41, NaCl 136., NaHCO3 4.17, Na2HPO4 0.34, sucrose 20, pH 7.4) and exposed to an oxygenfree environment (90% nitrogen, 5% hydrogen, and 5% CO2, 37°C) using a COY anaerobic
chamber (COY Laboratory Products Inc, Lake Charter Township, MI). OGD was terminated by
placing the cells back into glucose-containing maintenance media and returning cultures to a 5%
CO2, 37°C incubator. Sham IPC was performed using similar number of washes and glucoseFree HBSS, except glucose (20 mmol/L) was substituted for sucrose and cells were placed back
into normoxic incubator conditions.
Western Blot
Cells were lysed in RIPA Buffer (20 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1 mmol/L
EDTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L
Na 3VO4 and 1 mmol/L PMSF). Protein concentration was determined by BCA protein assay and
30 µg of protein was loaded onto a 12% SDS-polyacrylamide gel and electroblotted to
nitrocellulose. Membranes were blocked in 5% dry milk/TBST and hybridized with primary
antibodies overnight at 4°C. Blots were probed with rabbit anti-Nrf2 (1:500, Santa Cruz
Biotechnology, Dallas, TX), rabbit anti-UCP2 (1:500, Calbiochem Inc.), rabbit anti-GAPDH
(1:10000, Cell Signaling Technology, Danvers, MA), mouse anti-β-actin (1:10000, Sigma),
rabbit anti-MnSOD (1:2000, Cell Signaling Technology) or goat anti-Lamin-B (1:1000, Cell
Signaling Technology). Membranes were washed with TBST followed by incubation with antimouse, anti-goat, or anti-rabbit HRP-conjugated secondary antibodies (1:5000, Pierce, Thermo
Scientific; Rockford, IL) for 1 hr at room temperature. Proteins were detected using enhanced
chemiluminescence (ECL) system (Pierce, Thermo Scientific). Western blot densitometry was
analyzed using ImageJ (NIH)6.
ELISA
Nuclear and cytoplasmic fractionation was performed according to manufacturer’s protocol by
using a nuclear extract kit (Active motif). Nuclear and cytoplasmic extracts were probed with
both Lamin B and GAPDH to establish purity of the nuclear and cytoplasmic fractions
respectively. 10 µg of nuclear samples were used for the TransAM Nrf2 ELISA kit (Active
Motif, catalogue# 50296) to measure DNA binding of activated Nrf2 nuclear protein, as
determined by absorbance measurements at 450 nm.
Animal model of Focal Cerebral Ischemia
For our focal cerebral ischemia model, the left middle cerebral artery (MCA) was occluded for 1
hr using a poly-d-lysine-coated 3-0 nylon monofilament as previously described7 in C57black/6
mice. To measure blood flow during the MCA occlusion (MCAO), a laser Doppler flowmetry
probe inserted in the temporal lobe to measure perfusion of the MCA territory. Regional cerebral
blood flow was measured over the middle cerebral artery territory via Laser Doppler flowmetry
(Perimed, Stockholm, Sweden). For lasser Doppler, a flexible 0.5-mm fiberoptic probe was
affixed to the exposed skull over the ischemic cortex at 2 mm posterior and 3 mm lateral to
bregma in mice. The suture was advanced into the external carotid artery and advanced through
the internal carotid artery until an approximately 70% decrease in blood flow to the middle
cerebral artery occurred as measured by LDF. 48 hrs prior to ischemia, animals of each strain
were randomized into treatment groups and a blinded investigator administered either agent
intraperitoneally (i.p.). C57Bl/6J WT or Nrf2-/- male mice between 7-11 weeks were used for
these experiments, and each group of mice were further separated into 2 different treatment
groups: resveratrol (10 mg/kg i.p., Sigma) or DMSO (Vehicle injection, i.p.). Following 1 hr of
occlusion, the filament was removed and the animal was returned to their cages. 24 hr following
reperfusion, mice were anesthetized and perfused with 0.9 % saline, followed by decapitation
and rapid brain removal. Brains were sliced into eight 1 mm thick coronal sections using a
mouse brain matrix (RBM-200C, Activational Systems, Ann Arbor, MI, U.S.A.). These sections
were immersed in 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) dissolved in isotonic
saline and incubated at 37º C in the dark for 7 minutes. The sections were transferred to buffered
10% formalin for fixation and scanned into an image analysis system (M4, St. Catherine,
Ontario, Canada). Infarct areas were traced at each level and volumes were computed using a
direct and indirect method that corrects for edema using ImageJ software as described
previously8,9. Blood flow during the ischemic phase of the MCAO injury was monitored by laser
Doppler flowmetry. Exclusion criteria for these studies included (1) greater than 30% of baseline
MCA cerebral blood flow persisting through the occlusion phase of the MCAO injury (as
measured by laser Doppler); and (2) lack of detectable infarct following TTC staining.
Polarography
Mitochondrial respiration studies were conducted as previously described10. In brief, nonsynaptic mitochondria were isolated from WT or Nrf2-/- mice treated with resveratrol (10 mg/kg
intraperitoneal injection, i.p.) or dimethyl sulfoxide (DMSO) vehicle. Mouse cortex were
homogenized in isolation medium (250 mM sucrose, 1 mg/ml bovine serum albumin (fraction V
essentially fatty acid free, BSA), 1.0 mM ethylenediaminetetra-acetic acid (EDTA), and 0.25
mM dithiothreitol, pH 7.4) Tissue was minced with a pair of scissors and rinsed thoroughly with
the isolation medium. The minced tissue was homogenized in a hand-operated Teflon glass
homogenizer by 7-8 strokes. The homogenate was diluted to yield 10% (w/v) homogenate and
centrifuged at 720 g for 5 min using a Sorvall (Newton, CT) RC5 centrifuge. The supernatant
was collected in another tube and centrifuged again at the same speed to reduce nuclear
contamination of the eventual mitochondria sample. To isolate glial and neuronal cell body
mitochondria, non-synaptic mitochondria was collected by layering the supernatant obtained
from the final slow-speed centrifuge on a 24% (v/v) percoll gradient (percoll diluted in isolation
media with BSA). The gradients were centrifuged at 32,500 g for 5 min. The resulting pellet was
washed once with isolation media and centrifuged at 15,000 g for 10 min. The pellet was again
washed with 0.25 M sucrose by centrifugation at 15,000 g for 10 min. The resulting pellet was
resuspended in 0.25 M sucrose, and protein content was determined by bicinchoninic acid (BCA)
assay. All mitochondrial isolation procedures were performed at 4°C. The rate of state III
mitochondrial oxygen consumption was determined using a Clark-type oxygen electrode in the
presence of 100 µg non-synaptic mitochondria, 5 mmol/L pyruvate, 2.5 mmol/L malate, and 5
mmol/L ADP (excess). To induce state IV respiration, 5 µmol/L oligomycin was added to the
polarographic chamber to inhibit ATP synthase and coupled respiration. Ratio of State III/State
IV respiration yielded the respiratory control index, or RCI, an established measure of
mitochondrial coupling11.
Measurement of mitochondria ROS production
Mitochondrial ROS production was determined using a spectrophotometer following a
previously modified protocol12. Isolated non-synaptic mitochondria from Nrf2-/- or C57Bl/6J
animals were added to a microplate. H2O2 emission was measured fluorescently at 555 nm
(excitation)/590 nm emission
wavelengths. After establishing baseline measurements,
respiratory substrates were added in a similar manner to the polarographic studies. The
production of H2O2 was determined spectrophotometrically, and the electron transport chain site
of this production could be determined based on the combination of substrates added. Rates of
H2O2 emission were recorded for each complex-specific substrate/inhibitor pair, and normalized
to baseline H2O2 production for each sampled well.
Statistical Analysis
All data are expressed as mean ± STDEV. Statistical analysis between two groups was
performed using the unpaired Student’s t-test. Statistical analysis between more than two groups
was performed using a one-way ANOVA with Bonferroni’s multiple comparison post hoc test
unless otherwise specified. p ≤ 0.05 was considered statistically significant.
Supplementary Figure I: Nrf2-/- mice present with reduced levels of the Nrf2-regulated
antioxidant protein NQO-1. A) Standard PCR genotyping of Nrf2-/- mice and astrocyte cultures.
Ear punch samples from Nrf2-/- mice were subjected to genotyping analysis. Using genomic
DNA, the 400-bp PCR product was detected only in Nrf2-/- compared to the 262-bp product
detected in WT mice. B) Western blot of whole brain cortical lysate from WT and Nrf2-/- mice.
NQO-1 proteins levels were measured using Western blotting, along with Actin protein (loading
control). n = 4-6.
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