Free Radical Biology & Medicine 43 (2007) 408 – 414

www.elsevier.com/locate/freeradbiomed

Original Contribution
Role of Nrf2 in protection against intracerebral hemorrhage injury in mice
Jian Wang a , Jocelyn Fields a , Chunying Zhao a , John Langer a , Rajesh K. Thimmulappa b ,
Thomas W. Kensler b , Masayuki Yamamoto c , Shyam Biswal b , Sylvain Doré a,d,⁎
a
Anesthesiology/Critical Care Medicine, Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA
b
Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA
c
Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan
d
Department of Neuroscience, Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA

Received 22 November 2006; revised 13 April 2007; accepted 19 April 2007

Available online 29 April 2007

Abstract

Nrf2 is a key transcriptional factor for antioxidant response element (ARE)-regulated genes. While its beneficial role has been described for
stroke, its contribution to intracerebral hemorrhage (ICH)-induced early brain injury remains to be determined. Using wild-type (WT) and Nrf2
knockout (Nrf2−/−) mice, the role of Nrf2 in ICH induced by intracerebral injection of collagenase was investigated. The results showed that injury
volume was significantly larger in Nrf2−/− mice than in WT controls 24 h after induction of ICH (P b 0.05), an outcome that correlated with
neurological deficits. This exacerbation of brain injury in Nrf2−/− mice was also associated with an increase in leukocyte infiltration, production of
reactive oxygen species, DNA damage, and cytochrome c release during the critical early phase of the post-ICH period. In combination, these
results suggest that Nrf2 reduces ICH-induced early brain injury, possibly by providing protection against leukocyte-mediated free radical oxidative
damage.
© 2007 Elsevier Inc. All rights reserved.

Keywords: DNA damage; Free radicals; Inflammation; NF-E2-related factor 2; Reactive oxygen species

Introduction (Nrf2), a key transcriptional factor for antioxidant response

element (ARE)-regulated genes [4].
Clinical and animal studies have provided evidence that Nrf2 is regarded as a protector for many organs, including
inflammation and oxidative stress from reactive oxygen species brain (reviewed in [5]). It has been reported that Nrf2, a key
(ROS) are involved in the progression of intracerebral hemor- regulator of cell survival [6,7], can induce and up-regulate
rhage (ICH)-induced early brain injury [1–3]. In addition, cytoprotective and antioxidant genes that attenuate tissue injury
recent research has demonstrated that oxidative stress can mo- [8,9]. Sulforaphane, a naturally occurring isothiocyanate that
dulate inflammatory responses during tissue injury, possibly induces the expression of multiple Nrf2-responsive genes, has
through activation of nuclear factor erythroid 2-related factor 2 been shown to be neuroprotective against focal cerebral ische-
mia in rats [10]. In addition, activation of the Nrf2 pathway,
either by sulforaphane itself or by Nrf2 overexpression, was
Abbreviations: 8-OHG, 8-hydroxyguanosine; ARE, antioxidant response
element; FJB, Fluoro-Jade B; GST, glutathione S-transferase; ICH, intracerebral
able to protect neurons from oxidative stress damage [11]. Fur-
hemorrhage; IR, immunoreactive; NQO1, NAD(P)H: quinone oxidoreductase 1; thermore, primary cultured neurons derived from Nrf2 knock-
Nrf2, nuclear factor erythroid 2-related factor; ONOO−, peroxynitrite; ROS, out (Nrf2−/−) mice were shown to be more vulnerable to
reactive oxygen species; WT, wild-type. oxidative stress than neurons from control animals. However,
⁎ Corresponding author. Departments of Anesthesiology/Critical Care Med-
when the neurons were transfected with a functional Nrf2
icine and Neuroscience, Johns Hopkins University, School of Medicine, 720
Rutland Ave., Ross 365, Baltimore, MD 21205, USA. Fax: +1 410 9557271. construct, they become more resistant to free radicals [12].
E-mail address: [email protected] (S. Doré). Consistent with the results of these studies, dominant-negative-
URL: http://www.hopkinsmedicine.org/dorelab (S. Doré). Nrf2 and siRNA-Nrf2-stable neuroblastoma cell lines were
0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2007.04.020
 J. Wang et al. / Free Radical Biology & Medicine 43 (2007) 408–414 409

more prone to apoptosis than cells transfected with vector only neurologic scoring system [21] 24 h after collagenase injection.
because of the down-regulation of ARE-mediated protective The tests included body symmetry, gait, climbing, circling
genes [13]. behavior, front limb symmetry, and compulsory circling. Each
Previous studies have shown that increasing Nrf2 activity test was graded from 0 to 4, establishing a maximum deficit
provides protection against cerebral ischemia in vivo [10,11,14], score of 24. Immediately after the testing, the mice were sacri-
but the role of Nrf2 activity during hemorrhage has not yet been ficed for injury analysis.
examined. In this study, we hypothesized that Nrf2 would be
protective in intracerebral hemorrhage. To test this hypothesis, we Hemorrhagic injury analysis
subjected wild-type (WT) and Nrf2−/− mice to an ICH model that
caused disruption of blood vessels and entry of blood into the brain All processing and analysis of tissue sections as described in
parenchyma [3].Thenwe compared the outcomesin terms ofbrain this and the following sections were conducted by an observer
injury volume, number of degenerating neurons, neurologic blind to the genotype of the mice. Nrf2−/− (n = 7/group) and WT
function, inflammatory response, and ROS production. (n = 10/group) mice were euthanized, and their brains were
harvested, fixed in 4% paraformaldehyde for 24 h, and
Materials and methods cryoprotected in serial phosphate-buffered sucrose solutions
(20, 30, and 40%) at 4°C. Then the brains were cut into 50-
Animals μm sections with a cryostat. Sections were stained with Luxol
fast blue and Cresyl Violet [20] before being quantified for
This study was conducted in accordance with the National injury area with SigmaScan Pro software (version 5.0.0 for
Institutes of Health guidelines for the use of experimental ani- Windows; Systat, Port Richmond, CA). Six to eight coronal
mals. Experimental protocols were approved by the Johns slices from different levels of the injured hemorrhagic area
Hopkins University Animal Care and Use Committee. Nrf2−/− were summed, and the volumes in cubic millimeters were
and WT mice on a CD1 background were generated as des- calculated by multiplying the thickness by the measured areas
cribed previously [15,16] and were maintained in our facilities. [20].
All mice were subjected to genotyping for Nrf2 status by PCR
amplification of genomic DNA extracted from tail tips [17]. Histology
Three primers were used to perform PCR amplification: 5′-
TGGACGGGACTATTGAAGGCTG-3′ (sense for both geno- Luxol fast blue/Cresyl Violet, and Fluoro-Jade B (FJB)
types), 5′-CGCCTTTTCAGTAGATGGAGG-3′ (antisense for staining were performed according to published protocols
WT mice), and 5′-GCGGATTGACCGTAATGGGATAGG-3′ [22,23]. Cells permeable to FJB are marked as degenerating
(antisense for LacZ). These CD1 mice were fed with an AIN- neurons. To perform the quantification analysis, three sections
76A diet, given water ad libitum, and housed under controlled per mouse with similar areas of hematoma were chosen from
conditions (23 ± 2°C; 12 h light/dark cycle). three WT and three Nrf2−/− mice with similar brain injury vo-
lumes, and positively stained cells were counted in four differ-
ICH model ent comparable fields adjacent to the hematoma. Three sections
per animal over a microscopic field of 40× were averaged and
The procedure for inducing ICH by collagenase injection in expressed as cells/field, as previously reported [20]. Stained
mice, adapted from an established rat protocol [18], has been sections were examined with a fluorescence microscope; the
described previously [19,20]. Age- and weight-matched adult images were captured and analyzed by SPOT image software
male mice (26–33 g) were anesthetized by intraperitoneal (Diagnostic Instruments Inc., Sterling Heights, MI). Areas with
injection with Avertin (2-2-2 tribromoethanol; Sigma, St. Louis, large blood vessels were avoided.
MO; 0.5 mg/g body weight). To induce hemorrhage, mice were
injected unilaterally into the caudate putamen with collagenase Immunofluorescence
VII-S (0.1 U in 500 nl saline, Sigma) at the following stereo-
tactic coordinates: 0.8 mm anterior and 2.5 mm lateral of the Immunofluorescence was carried out as described previously
bregma, 2.5 mm in depth. Collagenase was delivered over [24]. Briefly, free-floating sections were washed in PBS for
5 min, and the needle was left in place for an additional 25 min 20 min, blocked in 5% normal goat serum, and incubated over-
to prevent any reflux. Rectal temperature was maintained at night at 4°C with primary antibodies: rabbit anti-myeloperox-
37.0 ± 0.5°C throughout the experimental and recovery periods. idase (MPO, neutrophil marker; 1:100; DAKO, UK); rabbit
Because the focus of our study was the early brain injury in anti-Iba 1 (microglia marker; 1:1000; Wako Chemicals,
ICH, mice were sacrificed for analysis 24 h later, after being Richmond, VA); mouse anti-nitrotyrosine (peroxynitrite mar-
tested for neurologic deficits. ker; 1:1000; Upstate, Lake Placid, NY); mouse anti-8-hydro-
xyguanosine (8-OHG; 10 μg/ml, Oxis International Inc, Port-
Neurologic deficit land, OR); mouse anti-cytochrome c (1:1000; BD Pharmingen,
San Diego, CA). To assess the cellular source of markers of
An experimenter blinded to the mouse genotype scored all oxidative stress (nitrotyrosine and 8-OHG) and cytochrome c
mice (10 WT; 7 Nrf2−/−) for neurologic deficits with a 24-point after ICH, double immunofluorescence was performed with one
410 J. Wang et al. / Free Radical Biology & Medicine 43 (2007) 408–414

Statistics

All data are expressed as means ± SD. Differences between

groups were determined by Student's t test. Statistical signi-
ficance was set at P b 0.05.

Results

Nrf2−/− mice have larger brain injury volumes and greater

neurologic deficit than WT mice after ICH

From previous in vitro and in vivo studies that demonstrated

a neuroprotective role for Nrf2, we hypothesized that Nrf2 gene
deletion would lead to increased brain injury after ICH. Quan-
tification of brain injury with Luxol fast blue/Cresyl Violet
staining confirmed that injury volume of Nrf2−/− mice (24.1 ±
7.4 mm3) was larger than that of WT mice (14.7 ± 4.4 mm3,
P = 0.015) 24 h after ICH (Figs. 1A and 1B). These results are
consistent with our previous studies [21,24]. No detectable
bleeding was observed in sham-operated mice (data not
shown).
To further determine whether the greater ICH-induced brain
injury in Nfr2−/− mice correlated with greater neurobehavioral
deficits, assessment of neurologic function of the animals was
performed at 24 h after collagenase injection. Nrf2−/− mice
showed more severe neurologic deficits than WT mice after
ICH (15.5 ± 4.0 vs 10.9 ± 1.9, P = 0.006) (Fig. 1C). The largest
differences in scores between Nrf2−/− and WT mice were in the
attributes of body symmetry, circling behavior, and compulsory

Fig. 1. Deletion of Nrf2 increases brain injury volume and neurologic deficits in
mice subjected to intracerebral hemorrhage (ICH). Age- and weight-matched Nrf2
knockout (Nrf2−/−) and wild-type (WT) mice were subjected to ICH, and brains
were sectioned and stained with Luxol fast blue/Cresyl Violet. (A) Representative
sections from Nrf2−/− and WT mice 24 h after collagenase injection showing
different areas of injury as represented by lack of staining. Scale bar = 100 μm. (B)
Quantification shows significantly larger brain injury volume in Nrf2−/− mice
(n = 7) compared with WT mice (n = 10) 24 h after collagenase injection. (C) An
investigator blinded to genotype assessed the neurologic deficits of Nrf2−/− and
WT mice with a 24-point neurologic scoring system 24 h after collagenase
injection. Neurologic deficits were significantly more severe in Nrf2−/− mice
(n = 7) than in WT mice (n = 10). Values are means ± SD; *P b 0.05.

of these markers and an antibody against microtubule-asso-

ciated protein-2 (MAP2, neuronal marker; 1:1000; Chemicon,
Temecula, CA). Sections then were incubated with Alexa488
(1:1000; Molecular Probes) and/or Cy3 (1:1000; Jackson
ImmunoResearch, West Grove, PA)-conjugated secondary anti-
body. Three sections per mouse with similar areas of hematoma
were chosen from WT and Nrf2−/− mice (three mice per group)
with similar brain injury volumes, and positively stained cells
were counted in four different comparable fields adjacent to the
hematoma. Three sections per animal over a microscopic field Fig. 2. Deletion of Nrf2 increases the number of degenerating neurons in mice
of 60× (for neutrophils) or 40× (for microglia/macrophages) subjected to ICH. (A) Fluoro-Jade B histological staining of degenerating
were averaged and expressed as cells/field. Stained sections neurons in sections collected 24 h after collagenase injection shows intensely
labeled neurons and processes in the peri-ICH region in WT and Nrf2−/− mice.
were examined with a fluorescence microscope as described Scale bar = 20 μm. (B) Quantification analysis suggested that Nrf2−/− mice had
above. Control sections were processed by the same method, more degenerating neurons than WT control mice, but the difference did not
except that primary antibodies were omitted. reach statistical significance (n = 3/group, P = 0.08). Values are means ± SD.
 J. Wang et al. / Free Radical Biology & Medicine 43 (2007) 408–414 411

had significantly more neutrophils (Fig. 3E, 39.2 ± 4.0 vs 30.4 ±

3.4 cells/field, n = 3/group, P = 0.04).
Microglial/macrophage activation contributes to ICH-
induced early brain injury [1–3]. To clarify the effect of Nrf2
on the state of microglial/macrophage activation after ICH, Iba1,
a marker for microglia/macrophages, was used [25]. The results
showed that resting microglial cells were sparse, but distributed
similarly in WT and Nrf2−/− mice on the uninjected side 24 h
after ICH (data not shown). Similarly, no differences were
apparent in the distribution of activated microglia/macrophages
around the injury site in WT and Nrf2−/− mice (Figs. 3C–3E).

Nrf2 deletion increases ROS production, DNA damage, and

cytochrome c release

ROS are thought to play a major role in the various me-

chanisms of ICH-induced brain injury [1,24]. Peroxynitrite
(ONOO−) is one of the ROS produced by the interaction of
nitric oxide (NO) and superoxide. ONOO− , acting as an oxi-
dant, is more stable than NO or superoxide and can readily
diffuse across phospholipid membranes [26]. We detected
ONOO−-positive cells around the injury site 24 h post-ICH in

Fig. 3. Deletion of Nrf2 increases leukocyte infiltration, but does not affect
microglial activation in mice subjected to ICH. (A–D) Infiltrating neutrophils
(MPO-positive cells; scale bar: 40 μm) and activated microglia (Iba1-positive
cells; scale bar: 20 μm) were apparent in or around the injury site in Nrf2−/− and
WT mice 24 h post-ICH. (E) Quantification analysis indicated that Nrf2−/− mice
had significantly more infiltrating neutrophils than WT mice at 24 h post-ICH;
the number of activated microglial cells around the injury site was similar in
Nrf2−/− and WT mice (both n = 3/group, *P b 0.05).

circling. We have previously observed that anesthesia alone has

no effect on the neurologic function of mice [21].
To examine whether neuronal death was more evident at the
site of hemorrhage in Nrf2−/− mice, we used FJB histological
staining, a specific marker for degenerating neurons [20,23].
The results suggest a trend toward more degenerating neurons
in Nrf2−/− than in WT mice (Fig. 2A), though they did not reach
statistical significance (32.7 ± 5.4 vs 23.2 ± 5.0 cells/field, n = 3/
group, P = 0.08) (Fig. 2B). FJB-positive neurons were not
observed in the contralateral side or normal brain, but were
occasionally observed along the needle track in sham-operated
WT and Nrf2−/− mice (data not shown).

Nrf2 deletion increases leukocyte infiltration Fig. 4. Deletion of Nrf2 increases ROS production in mice subjected to ICH.
Peroxynitrite (ONOO−) was used as a marker for ROS production. (A) Increased
Acute inflammation is a normal response to brain injury. As ONOO− immunoreactivity (IR) was detected in the cytosol of cells around the
indicated by immunoreactive MPO, ICH produces a robust injury site 24 h post-ICH in tissue sections from WT and Nrf2−/− mice. Scale
infiltration of neutrophils into the affected striatum that can be bar = 20 μm. (B) Double labeling of nitrotyrosine and MAP2 in WT mice indi-
cated that nearly all the ONOO−-positive cells were neurons. Scale bar = 30 μm.
observed as early as 4 h after ICH [24]. Although infiltrating (C) Quantification of ONOO–-immunopositive cells around the injury border
neutrophils were evident in and around the injury site in WT and region showed that Nrf2−/− mice had significantly more positive cells than WT
Nrf2−/− mice 24 h post-ICH (Figs. 3A and 3B), Nrf2−/− mice mice (n = 3/group, *P b 0.05). Values are the means ± SD.
412 J. Wang et al. / Free Radical Biology & Medicine 43 (2007) 408–414

WT and Nrf2−/− mice as indicated by the ONOO− staining in

Fig. 4A. We did not detect ONOO–-positive cells on the
contralateral side or in normal brain. Double labeling of
nitrotyrosine and MAP2 demonstrated that nearly all the
ONOO–-positive cells in WT mice were neurons (Fig. 4B).
Quantification analysis showed that Nrf2−/− mice had sig-
nificantly more ONOO–-positive cells than WT mice around
the border region of injury (Fig. 4C, 17.0 ± 1.7 vs 12.6 ± 1.5
cells/field, n = 3/group, P = 0.03). ONOO–-positive cells were
observed very rarely in sham-operated WT and Nrf2−/− mice
(data not shown).
8-Hydroxyguanosine is a reliable and commonly used bio-
marker for oxidative DNA damage caused by superoxide anion,
as shown previously after various forms of brain injury [27–29],
including ICH [28,30]. Here, 8-OHG-positive cells were
detected around the injury site 24 h post-ICH in WT and
Nrf2−/− mice (Fig. 5A). Double labeling of 8-OHG and MAP2
demonstrated that nearly all the 8-OHG-positive cells in WT
mice were neurons (Fig. 5B). Analysis showed that Nrf2−/−
mice had more 8-OHG-positive cells than WT mice around the
border region of injury at 24 h post-ICH (Fig. 5C, 19.3 ± 2.6 vs
9.2 ± 2.0 cells/field, n = 3/group, P = 0.006). 8-OHG-positive

Fig. 6. Deletion of Nrf2 increases cytochrome c release in mice subjected to

ICH. (A) Increased cytochrome c immunoreactivity (IR) was detected in the
cytosol of cells around the injury site 24 h post-ICH in WT and Nrf2−/− mice.
Scale bar = 40 μm. (B) Double labeling of cytochrome c and MAP2 in WT mice
indicated that most cytochrome c-immunopositive cells were neurons. Scale
bar = 30 μm. (C) Quantification of cytochrome c-positive cells around the injury
border region showed that Nrf2−/− mice had significantly more positive cells
than WT mice (n = 3/group, *P b 0.05). Values are means ± SD.

cells were not observed in the contralateral side or in normal

brain, but were observed very rarely in sham-operated WT and
Nrf2−/− mice (data not shown).
To understand the mechanisms implicated in cell death after
ICH, we examined the immunoreactive cytosolic cytochrome c.
Release of mitochondrial cytochrome c to the cytosol has been
linked to apoptotic cell death [31], a significant contributor to
ICH-induced brain damage [20,32,33]. We did not detect cyto-
chrome c in the control hemisphere. In contrast, we did detect
cytochrome c immunoreactivity in the cytosol of the cells around
the border region of injury 24 h post-ICH (Fig. 6A). Double
labeling of cytochrome c and MAP2 in WT mice demonstrated
that most cytochrome c-positive cells were neurons (Fig. 6B).
Analysis suggested that around the border region of injury,
Nrf2−/− mice had more cytochrome c-positive cells than WT
mice (Fig. 6C, 23.1 ± 4.2 vs 11.2 ± 0.8 cells/field, n = 3/group, P =
Fig. 5. Deletion of Nrf2 increases DNA damage in mice subjected to ICH. 8- 0.03). Cytochrome c-positive cells were observed very rarely in
Hydroxyguanosine (8-OHG) was used as a marker for DNA oxidation. (A) sham-operated WT and Nrf2−/− mice (data not shown).
Increased 8-OHG immunoreactivity (IR) was detected in the cytosol of cells
around the injury site 24 h post-ICH in tissue sections from WT and Nrf2−/− Discussion
mice. Scale bar = 20 μm. (B) Double labeling of 8-OHG and MAP2 in WT mice
indicated that nearly all the 8-OHG-immunopositive cells were neurons. Scale
bar = 30 μm. (C) Quantification of 8-OHG-positive cells around the injury This study revealed that Nrf2−/− mice are significantly more
border region showed that Nrf2−/− mice had significantly more positive cells prone to hemorrhagic brain injury and neurologic deficits than
than WT mice (n = 3/group, *P b 0.01). Values are means ± SD. their WT counterparts. Furthermore, we found that Nrf2−/− mice
 J. Wang et al. / Free Radical Biology & Medicine 43 (2007) 408–414 413

have more neuronal cell death, neutrophil infiltration, ROS and also suggest that Nrf2 deficiency could enhance ICH-
production, DNA damage, and cytochrome c release. Although induced neuronal cell death possibly by apoptotic mechanisms.
previous work has shown that after a permanent stroke model One potential defense against the toxicity of oxidative stress
(permanent middle cerebral artery occlusion without reperfu- is the induction of a family of phase II detoxification enzymes.
sion) Nrf2−/− mice suffered more stroke damage than WT ARE, a unique cis-acting regulatory sequence, is essential for
controls [34], a finding that we have confirmed and extended in the constitutive and induced expression of many antioxidant
a transient stroke model (middle cerebral artery occlusion with genes involved in the phase II pathway [46]. Available evidence
reperfusion) [35]; to our knowledge, these findings reported suggests that Nrf2 is a major transcription factor responsible for
here provide the first clear evidence that Nfr2 plays a critical upregulating ARE-mediated antioxidant gene expression such
role in limiting the cascade of events leading to ICH-induced as NAD(P)H: quinone oxidoreductase 1 (NQO1), glutathione
early brain injury. S-transferase (GST), heme oxygenase 1 (HO-1), glutamylcys-
Oxidative stress from ROS contributes to ICH-induced early teine ligase (the rate-limiting enzyme in glutathione synthesis),
brain injury [1–3]. In this study, ONOO− (a marker for ROS) thioredoxin, and thioredoxin reductase 1 [4,12,47]. Basal
and 8-OHG (a marker for DNA oxidation) were found mostly in NQO1 and GST activities were found to be lower in multiple
neurons bordering the injury site at 24 h post-ICH; however, brain regions of Nrf2−/− mice, compared with WT mice [34].
more ONOO−- and 8-OHG-positive neurons were observed in Therefore, it is likely that deletion of the Nrf2 gene renders mice
Nrf2−/− mice than in WT mice. It is therefore likely that the more susceptible to ICH-induced early brain injury because of
exacerbated injury from hemorrhage observed in Nrf2−/− mice decreased ability to induce phase II detoxification enzymes.
is, at least in part, attributable to the increase in post-ICH ROS Additional work is necessary to determine which ones or which
production. combinations are responsible for the beneficial effect of Nrf2-
After brain injury, activated leukocytes and microglia/ mediated gene expression.
macrophages are major sources of ROS production [2,36–38], In conclusion, we have shown that Nrf2-deficient mice are
and available data from clinical and preclinical animal models significantly more susceptible to ICH-induced early brain injury
support a role for activation of leukocytes and microglia/ than control mice. The exacerbation of injury appears to be
macrophages in ICH-induced early brain injury [24,39–41]. In associated with an increase in leukocyte infiltration, ROS
our study, more infiltrating neutrophils were observed in Nrf2−/− production, DNA damage, and cytochrome c release during the
mice at 24 h post-ICH, but no difference in microglia/macro- critical phase of the early post-ICH period. Taken together, these
phage activation was found between WT and Nrf2−/− mice at the results suggest that Nrf2 deficiency contributes to ROS-induced
same time point. These findings indicate that neutrophil infil- DNA damage and apoptosis mostly in neurons in the early stage
tration, rather than microglial/macrophage activation, correlates of ICH, and that activation of Nrf2 will serve to control the
with increased early brain injury in Nrf2−/− mice. Infiltrating infiltration of leukocytes into the focus of the injury, preventing
leukocytes damage brain tissue by increasing vascular perme- excessive free radical oxidative damage in the brain tissue.
ability, releasing proinflammatory proteases, and generating Although additional work with selective Nrf2 inducers and
ROS [24,42], which increase free radical oxidative damage in inhibitors is needed, the findings raise the possibility that Nrf2
neurons. Therefore, increasing Nrf2 activity in the early stage of will be a potential therapeutic target for the treatment of ICH.
ICH may diminish additional recruitment of leukocytes and
decrease leukocyte-mediated early brain injury. Acknowledgments
Human and animal studies have provided evidence that
apoptosis is a prominent form of cell death associated with ICH This work was supported by an American Heart Association
in the peri-hematoma region [20,33,43]. Using FJB staining as a SDG 0630223N (J.W.); NIH Grants HL081205 and P30ES0389
marker for neuronal death, we observed a trend toward more (SB); and AT001836, AA014911, AT002113, and NS046400
degenerating neurons in Nrf2−/− mice than in WT mice at 24 h (S.D.). We thank Claire Levine for assistance with the
post-ICH, although the difference was not statistically different. manuscript and all members of the Doré lab for their insightful
Oxidative stress from ROS has been shown to trigger cyto- comments.
chrome c release, which is often followed by DNA damage and
cell death [44]. To explore further whether Nrf2 deficiency
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