Article

Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury. J Neuroinflammation 7:41

Department of Emergency and Critical Care Medicine, Showa University School of Medicine, Shinagawa-Ku, Tokyo 142-8555, Japan.
Journal of Neuroinflammation (Impact Factor: 5.41). 07/2010; 7(1):41. DOI: 10.1186/1742-2094-7-41
Source: PubMed
ABSTRACT
We hypothesized that gp91phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O2-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation.
Unilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox after TBI were investigated using immunoblotting and staining techniques. Levels of O2- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNgamma or IL-4.
Gp91phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O2- and peroxynitrite metabolites produced were less in gp91phox-/- mice than in Wt. In the presence of IFNgamma, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox.
Classical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.

Full-text

Available from: Tomoya Nakamachi
RESEARC H Open Access
Gp91
phox
(NOX2) in classically activated microglia
exacerbates traumatic brain injury
Kenji Dohi
1*
, Hirokazu Ohtaki
2
, Tomoya Nakamachi
2
, Sachiko Yofu
2
, Kazue Satoh
2
, Kazuyuki Miyamoto
1
,
Dandan Song
2
, Shohko Tsunawaki
3
, Seiji Shioda
2
, Tohru Aruga
1
Abstract
Background: We hypothesized that gp91
phox
(NOX2), a subunit of NADPH oxidase, generates superoxide anion
(O
2
-
) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91
phox
and
reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91
phox
knockout mice
(gp91
phox-/-
). We also used a microglial cell line to determine the activated cell phenotype that contributes to
gp91
phox
generation.
Methods: Unilateral TBI was induced in gp91
phox-/-
and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression
and roles of gp91
phox
after TBI wer e investigated using immunoblotting and staining techniques. Levels of O
2
-
and
peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed
gp91
phox
was determined in a microglial cell line, BV-2, in the presence of IFNg or IL-4.
Results: Gp91
phox
expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of
Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O
2
-
and peroxynitrite
metabolites produced were less in gp91
phox-/-
mice than in Wt. In the prese nce of IFNg, BV-2 cells had increased
inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and
drastically increased expression of gp91
phox
.
Conclusions: Classical activated microglia promote ROS formation through gp91
phox
and have an important role in
brain damage following TBI. Modulating gp91
phox
and gp91
phox
-derived ROS may provide a new therapeutic
strategy in combating post-traumatic brain injury.
Background
Traumatic brain injury (TBI) is a serious condition in
emergency medicine, and its pathophysiological profile
is varied and complicated. One of the neurotoxic factor s
thought to be involved is o xidative stress [1,2]. A lar ge
number of studies have reported that oxidative stress,
which generates reactive o xygen species (ROS), plays a
key role in the development of TBI [1,3,4]. Conse-
quently, one of the most obvious ways to manage T BI
may be to control ROS generation [1] g iven that animal
experiments have supported the notion that free radical
scavengers and antioxidants dramatically reduce cerebral
damage [1,5,6]. The superoxide anion (O
2
-
) is an impor-
tant free radical, and is the source of other ROS that
lead to li pid peroxidation [7]. Cyclooxygenase, xanthine
oxidase, and NADPH oxidases of the NOX family are
well known generators of O
2
-
in the brain. However, the
main cellular mediator of O
2
-
generation after TBI has
not yet been determined. NADPH oxidase, a multiunit
enzyme initially discovered in neutrophils, has recently
emerged as a major generator of ROS in neurons, glial
cells and cerebral blood vessels [8-10]. NADPH oxidase
is composed of membrane-bound (p22
phox
and
gp91
phox
) and cytoplasmic subunits (p40
phox
,p47
phox
,
and p67
phox
). Several homolo gs of the catalytic subunit
of the enzyme, gp91
phox
, also termed N OX2, exist
(NOX1 through NOX5) [11,12]. It has been reported
that gp91
phox
-containing NADPH oxidase produces a
large amount of O
2
-
in leukocytes, while numerous
papers have reported on the role for gp91
phox
in various
neurodegenerative conditions [13,14]. However, the
source and the roles of gp91
phox
after TBI have not
* Correspondence: kdop@med.showa-u.ac.jp
1
Department of Emergency and Critical Care Medicine, Showa University
School of Medicine, Shinagawa-Ku, Tokyo 142-8555, Japan
Dohi et al. Journal of Neuroinflammation 2010, 7:41
http://www.jneuroinflammation.com/content/7/1/41
JOURNAL OF
NEUROINFLAMMATION
© 2010 Dohi et al; licensee BioMed Central Ltd. This is an Open Access arti cle distributed u nder the terms of the Creative Common s
Attribution License (http://creativecommons.org/lice nses/by/2.0), which permits unrestricted us e, distr ibution, and reproduction in
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Page 1
been established. In this study, we used the gp91
phox-/-
mouse to investigate the kinetics and the roles of
gp91
phox
following TBI.
Methods
Animals
All experimental procedures involving animals were
approved by the Institutional Animal Care and Use
Committee of Showa University. The gp91
phox-/-
(C57/
B6J) mice are described by Dinauer et al. [15], Wild
mice (Wt) were generated from the same chimeric foun-
der, and experiments were performed in age- and
weight-matched animals.
Controlled cortical impact model
Mice were anesthetized with 2% sevoflurane in 70%
N
2
Oand30%O
2
. A controlled cortical impact was
made using a pneumatic ally controlled impactor device
as described previously [16].
Cell culture
We used the BV-2 microglial cell line to investigate
which microglia cells express gp91
phox
[17]. This m ouse
BV-2 cell line w as obtained from Interlab Cell Line Col-
lection (Genova, Italy) and cultured in 10% RPMI1640
(RPMI1640 with 10% fetal calf serum [FCS], 100 U/ml
penicillin, 100 μg/ml streptomycin, and 2 mM L-gluta-
mine [all from GIBCO/BRL, Grand Island, NY]). The
cells were grown at 37°C in a humidified 5% CO
2
incuba-
tor. After harvesting, the cells were washed with PBS
twice and resuspended with experimental medium (Dul-
beccos modified E agles medium [GIBCO/ BRL] with 1%
FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2
mM L-glutamine). The cells were seeded into six-well
plates at 1 × 10
6
cells/well/ml and then exposed to IFNg
(10 ng/ml), IL-4 (20 ng/ml), IL-10 (10 ng/ml, all from
Peprotech, Rocky Hill, NJ), or vehicle (n = 3/group).
Twenty-four hours later, the cells were collected by cen-
trifugation. The samples were kept at -30°C until analysis.
Western blot analysis
The cerebrum was removed from decapitated animals at
0 (sham-operated), 24, and 48 hours after TBI and
divided into the ipsilateral and co ntralateral hemi-
spheres. These samples were then homogenized in lysis
buffer (10 mM Tris-HCl [pH 7.4], 0. 15 M NaCl and 1%
Triton X-100, 1 mM EGTA, 50 mM NaF, 2 mM sodium
orthovanadate, 10 mM sodium pyrvate, and protease
inhibitor cocktail [Sigma, St. Louis, MO]), and centri-
fuged at 12,000 × g for 10 minutes on ice. BV-2 samples
were sonicated for 10 seconds with lysis buffer to pre-
pare cell suspensions.
After determination of the protein concentration (BCA
protein assay, Thermo Fisher Scientific, W altham, MA),
appropriate amounts of samples were electrophoresed.
The separated proteins were then transferred to polyviny-
lidinene f luoride membranes (Bio-Rad, Hercules, CA).
After blocking with 2% Blockace (DS Pharma, Osaka,
Japan), the membranes were probed with primary antibo-
dies. After washing, the membrane w as probe d with
horseradish peroxidase (HRP)-conjugated secondary anti-
bodies. The protein bands were detected by chemilumi-
nescence (SuperSignal West Dura Extended Duration
Substrate; Pierc e, R ockfor d, IL) and exposed onto X-ray
film. The films were scanned, and the signal densities
were quantified using the UN-SCAN-IT gel analysis
program (Silk Scientific, Orem, UT).
Immunohistochemistry
Mice subjected to TBI were placed under pentobarbital
(50 mg/kg, i.p.) anesthesia and perfused with 0.9% NaCl
followed by 2% paraformaldehyde (PFA). Brains were
removed and processed to frozen blocks that were then
cut into 8-μm sections (n = 4-5/group).
The sections were incubated with 0.3% H
2
O
2
and then
incubated with PBS containing 5% normal horse serum
to mask nonspecific reactions. Next, the sections were
incubated with antibody r aised against gp91
phox
[18].
Onedaylater,thesectionswererinsedandincubated
with biotinylated goat anti-rabbit IgG (Santa Cruz
Biotechnology, Santa Cruz, CA), and then with an avi-
din-biotin complex solution (Vector Laboratories,
Burlingame, CA) followed by diaminobenzidine (DAB;
Vector) as a chromogen.
A similar procedure was used for multiple immunos-
taining, except that the sections were not incubated
with 0.3% H
2
O
2
, and were incubated with Alexa-labeled
fluorescence secondary antibodies. Primary and second-
ary antibodies for multiple-staining are listed in Table 1.
4,6-Diamidine-2-phenylindole dihydrochloride (DAPI,
1:10,000; Roche, Mannheim, Germany) was used for
nuclear staining. The fluoresc ence and immunolabeling
were detected using a confocal laser microscope (AX-10,
Zeiss; Oberkochen, Germany).
Evaluation of the injured brain area
The areas of injured brain were determined using 2,3,5-
triphenyltetrazolium chloride (TTC) staining of tissues
48 hours after TBI. The animals were decapitated, and
the brain was sectioned into four 2-mm coronal sections
by using a mouse brain matrix. The brain slices were
then stained with 2% TTC at 37°C for 30 min and
photographed on the anterior surface of each section
with a scale bar. The areas of injured brain were deli-
neated by examining differences betw een the ipsilateral
and contralateral regions in the center slice of i njured
brain and measured by using NIH Image software.
http://rsb.info.nih.gov/nih-image/about.html.
Dohi et al. Journal of Neuroinflammation 2010, 7:41
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Page 2
Evaluation of apoptosis-like cell death
To determine n eural apoptosis-like cell death, terminal
deoxynucleotidyl transferase-mediated dUTP end-label-
ing (TUNEL) staining (In Situ Cell Death Detection Kit,
POD; Roche) was performed 48 hours after TBI (n =5)
and the number of TUNEL-positive cells in the ipsilat-
eral hemisphere was then counted and compared
gp91
phox-/-
with W t mice in a similar co rtical region
(40 × magnification).
In situ detection of O
2
-
Produ ction of O
2
-
was determined by in situ detection of
oxidized hydroethidium (HEt) [19]. With the animal
placed under anesthesia, the HEt solution wa s adminis-
tered (1 mg/mL 0.9% NaCl with 1% DMSO) into the
jugular vein (n = 3 per group) 48 ho urs after TBI. Fifteen
minutes later, the brain was removed and frozen in
blocks and cryosectioned (8 μm) in the coronal plane. To
demonstrate the cellular distribution of Et, the sections
were co-stained with antibodies raised against gp91
phox
,
CD11b, GFAP, or NeuN. Fluo rescence was detected
using confocal laser microscopy (AX-10, Zeiss, Germany)
NO and TNFa measurement in media
Levels of NO and TNFa production are markers of clas-
sically activated microglia [20 ]. NO production was mea-
sured using the Griess method (Dojindo, Kumam oto,
Japan) as total NO (NO
2
-
and NO
3
-
). TNFa production
was measured by enzyme-linked immunosorbent assay
using the Duoset ELISA Development System (R&D
Systems, Minneapolis, MN).
Assay for arginase activity
Arginase is a marker for al ternatively activated microglia
[20]. Arginase activity was measured according to a pre-
vious paper [21] with minor modification . In brief, the
cell homogenate was mixed with equal volumes of pre-
warmed 50 mM Tris-HCl, pH 7.5 containing 10 mM
MnCl
2
and incu bated for 15 minutes at 55°C. The mix-
ture was incubated in 0.25 M L-arginine fo r 60 minutes
at 37°C to produce urea from arginine and the reactions
were stopped by adding Stop solution (H
2
SO
4
/H
3
PO
4
/
H
2
O, 1:3:7). Then, 1% (final concentration) 1-phenyl-1,
2-propanedione-2-o xime (ISPF) in ethanol was ad ded to
the solution, which was heated at 100°C for 45 min. The
reaction between urea and ISPF produc ed a pink color,
and absorption was measured at 540 nm.
Statistical analysis
Data are expressed as mean ± SE for in vivo experi-
ments. Data are expressed as m ean ± SD for in vitro
experiments S tatistical comparisons were performed
using the Students t tests and two-way analysis of var-
iance (ANOVA) as appropriate. P values less than 0.05
were considered statistically significant.
Results
Gp91
phox
is upregulated in the peri-contusional region
after traumatic brain injury
Fig 1 shows the results of immunoblotting experiments
to describe the expression of gp91
phox
after TBI. Protein
levels of gp91
phox
were not increased after TBI in the
contralateral hemisphere of Wt mice. In the ipsilateral
Table 1 Antibodies used for immunoblotting (IB) and immunohistochemistry (IHC)
Antibody Antigen Host Company Catalog # Dilution
Primary antibody (clone #)
gp91
phox
Human gp91 Rabbit See ref 18 4,000 (IB)
200 (IHC)
p22
phox
Human p22 Rabbit See ref 18 3,000
iNOS Mouse iNOS Rabbit Transduction Laboratories (Lexington, KY) N32030 10,000
Ym1 Mouse Ym1 Rabbit StemCell Tech (Vancouver, BC, Canada) 01404 1,000
GAPDH (6C5) Rabbit GAPDH Mouse Chemicon International (Temecula, CA) MAB374 3,000
b-Actin (AC-74) Mouse b-Actin Mouse Sigma (St Louise, MO) A5316 4,000
CD11b (5C6) Mouse CD11b Rat Serotec (Oxford, UK) MCA711 500
GFAP (G-A-5) Mouse GFAP Mouse Sigma (St Louise, MO) G3893 1000
NeuN Mouse NeuN Mouse Chemicon International (Temecula, CA) MAB377 1000
3-NT Nitrated KLH Rabbit Upstate Biotechnology (Lake Placid, NY) 06-284 100
Secondary antibody (conjugation)
Mouse IgG (HRP) Mouse IgG Sheep GE Healthcare Bioscience (Little Chalfont, UK) NA931 2,000
Rabbit IgG (HRP) Rabbit IgG Donkey GE Healthcare Bioscience (Little Chalfont, UK) NA934 3,000
Rabbit IgG (biotinylated) Rabbit IgG Goat Santa Cruz Biotechnology (Santa Cruz, CA) SC-2040 200
Mouse IgG (Alexa 546) Mouse IgG Goat Molecular Probes (Eugene, OR) A11030 400
Rabbit IgG (Alexa 488 or 546) Rabbit IgG Goat Molecular Probes (Eugene, OR) A11034 or 11035 400
Rat IgG (Alexa 546) Rat IgG Goat Molecular Probes (Eugene, OR) A11081 400
Dohi et al. Journal of Neuroinflammation 2010, 7:41
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Page 3
hemisphere of Wt mice, protein levels of gp91
phox
were
greater at 1 and 2 days after TBI relative to the contral-
ateral hemisphere (Fig 1A, B). The data indicate that
gp91
phox
increased in the ipsilateral hemisphere follow-
ing TBI stress.
The results of single immunohistochemical detection
of gp91
phox
after TBI are presented Fig 2A. Two days
after TBI, gp91
phox
immunoreactivity was dramatically
increased in the peri-contusion region.
Gp91
phox
is mainly expressed by cytotoxic-type classically
activated microglia in the peri-contusional regions after
TBI
To identify the cell types expressing gp91
phox
in the
peri-contusional area, cells were co-labelled with antibo-
dies raised against gp91
phox
and markers of microglial
(CD11b), astroglial (GFAP), and neuronal (NeuN) cells
(Fig 2B). The immunoreactivity for gp91
phox
was mainly
co-localized to microglia that had amoeboid-like mor-
phological features with round cell body and short pro-
cesses, suggesting cytotoxic-t ype classic al acti vation.
Immunoreactive s taining for gp91
phox
was also co-loca-
lized to a few astrocytes and neurons.
Gp91
phox
inhibition reduces the severity of TBI in vivo
TTC staining was used to evaluate the role of gp91
phox
on the severity of TBI in gp91
phox-/-
and Wt mice
(Fig 3A, B). Images of the TTC-stained anterior surface
of coronal sections demonstrated that the injured brain
area in gp91
phox-/-
mice was significantly smaller (P <
0.01) than that of the Wt mice (Fig. 3B). We verified the
prevention of the c ortical injury with TU NEL staining,
which was used to identify apoptotic-like cell death at
48 hours after TBI (Fig 3C, D). The number of TUNEL-
positive cells of gp91
phox-/-
mice was signi ficantly less
(p < 0.05) compared with Wt mice (Fig 3D).
Gp91
phox
gene deletion reduces superoxide radical (O
2
-
)
production after TBI
High intensity fluorescence staining by the oxidation
product Et was observed, indicating high levels of O
2
-
production. The peri-contusional area of Wt mice
accounted for much of the Et signal after TBI ( Fig 4A).
Et fluoresc ence w as lower in gp91
phox-/-
mice (Fig 4A)
compared to Wt mice (p < 0.05) (Fig 4B). The Et fluor-
escence was associated with p91
phox
-positive cells (Fig.
4C), and CD11b-positive microglial cells (Fig. 4D). Some
Figure 1 Characterization of gp91
phox
after traumat ic brain injury (TBI) using western immunoblots. (A) Immunoblotting signals before
and after TBI in wild-type (Wt) mice (left) and gp91
phox-/-
mice (right). (B) In Wt mice, gp91
phox
levels were significantly increased on day 1 and
day 2 after TBI (*p < 0.05 relative to sham). Gp91
phox
levels on the ipsilateral side were greater than on the contralateral side on day 1 and day 2
after TBI (*p < 0.05). Each value is the mean ± SE (n = 3). Note that the intensity of each sample was quantified and corrected relative to the
labeling control, actin. Increasing levels of gp91
phox
were not seen in gp91
phox-/-
mice.
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Page 4
Et fluorescence was slightly detected in a few neurons
(Fig.4D).TheseresultssuggestthatO
2
-
is mainly
produced by microglia expressing gp91
phox
.
Gp91
phox
gene deletion reduces 3-NT generation after TBI
in vivo
Peroxynitrite (ONOO
-
) is an oxidant and nitrating ag ent
and is synthesized by the reaction between O
2
-
and
nitric oxide (NO). ONOO
-
candamageawidearrayof
cellular molecules, including DNA and proteins. 3-NT is
an oxidized metabolite of ONOO
-
in vivo.Weper-
formed multiple-immunostaining for 3-NT and cell
marker antibodies in the peri-contusional area at
48 hours. Immunoreactivity for 3-NT in Wt mice clearly
co-localized with microglia, astrocytes, and degenerated
neurons (Fig 5; upper panel). In gp91
phox-/-
mice,
Figure 2 Expressions and cell identification of gp91
phox
in peri-contusional area after traumatic brain injury (TBI). (A) In sham-operated
animals, weak immunoreactivity for gp91
phox
was observed in the cortex (upper panel). Two days after TBI, gp91
phox
immunoreactivity was
dramatically increased in the peri-contusional area (lower panel). Scale bars = 400 μm. (B) Co-immunostaining of gp91
phox
with cell markers in
the peri-contusional region. Immunostaining was carried out using antibodies for gp91
phox
(shown in green) together with Integrin alpha M
(CD11b) (upper), glial fibrillary acidic protein (GFAP) (middle), and neuronal nuclear antigen (NeuN) (lower). Microglia, astrocyte, and neuron
markers are shown in red. Gp91
phox
immunoreactive cells were co-labeled with all cell markers. Particularly strong expression of gp91
phox
was
detected in microglial-like cells (CD11b-positive cells). Cells were counter-stained with DAPI to show nuclei (blue). Scale bars = 20 μm.
Dohi et al. Journal of Neuroinflammation 2010, 7:41
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Page 5
however, 3-NT immunoreactions were dramatically sup-
pressed in the microglial and astroglial cells (Fig 5).
While 3-NT immunoreactivity was observed in
gp91
phox-/-
mouse neurons, the integrity of these cells
was preserved (Fig 5). Taken together these findings
suggest that production of O
2
-
and ONOO
-
by gp91
phox
may influence the severity of TBI.
Classically activated BV-2 increased gp91
phox
and p22
phox
From the results presented in Fig. 2 we suggested that
gp91
phox
co-localized with classical activated microglia.
We then tried to identify the microglia that expressed
gp91
phox
by using the mouse microglial cell line BV-2
and by activating these cells with IFNg,IL-4,orIL-10.
As shown in Fig 6, the media from the BV-2 cells
Figure 3 Brain damage and cell death after traumatic brain injury in wild type (Wt) mice and gp91
phox-/-
mice. (A) 2,3,5-
triphenyltetrazolium chloride (TTC)-stained coronal brain sections from Wt mice (left) and gp91
phox-/-
mice (right) 2 days after TBI. (B) The
contusion area in gp91
phox-/-
mice (170.5 ± 71.5 mm
2
×10
-2
, n = 20) was significantly smaller than that in the Wt group (290.7 ± 94.0 mm
2
×
10
-2
, n = 10, *p < 0.01, t test). (C) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-staining in peri-contusional area of wild
type (left) and gp91
phox-/-
(right) mice 48 hours after TBI. TUNEL-positive cells numbers in the gp91
phox-/-
mice (117 ± 69.3 cells/area, n = 6) were
significantly smaller than those in the Wt group (252 ± 128.9 cells/area, n = 6, *p < 0.05, t test) (D). All values represent the mean ± SE.
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Page 6
showed significantly increased total NO after exposure
to IFN g, but not to IL-4 or IL-10 at 24 hours. The
IFNg-exposed BV-2 cells had significantly increased
TNFa in th eir media and expressed iNOS as well. All of
these factors indicate that the BV-2 cells were activated
according to the classically activated phenotype [20]. On
the other hand, the IL-4-exposed BV-2 cells showed
increased arginase activity and levels of Ym-1, indicating
an alternatively activated phenotype [20]. IL-10-exp osed
BV-2 cells did not show an y changes in levels of these
factors. Immunoblotting experiments showed that
gp91
phox
levels increased in the IFNg-exposed BV-2.
Moreover, immunoblotting for a different NADPH oxi-
dase subunit, p22
phox
, mirrored that o f gp91
phox
,sug-
gesting that not only gp91
phox
but also NADPH oxidase
expression was induced in the classically activated
microglia.
Discussions
We demonstrate h ere that gp91
phox
is increased in the
ipsilateral hemispher e after TBI and specifically in
amoeboid-shaped microglial cells. Mice that are gene-
deficient for gp91
phox
exhibit reduced primary cortical
damage, as evidenced by reduced areas of contusion,
Figure 4 The roles of gp91
phox
in supero xide radical production after traumatic brain inju ry). (A) Superoxide radical production
(hydroethidium [HEt]-positive cells: shown in red) in the peri-contusional area of wild type (upper) and gp91
phox-/-
(lower) mice 2 days after TBI.
(B) TBI-induced superoxide radical production (HEt-positive cells) in the peri-contusional area was significantly attenuated by gp91
phox
deficiency
in sham-operated mice, and 2 days after TBI (*p < 0.05, t test). All values represent mean ± SE. (C, D) Co-localization of HEt-positivity with cell
markers in the peri-contusional region. Cell identification was carried out using antibodies for gp91
phox
(C), CD11b (D, left), GFAP (D, middle), and
Neu N (D, right) (shown in green). HEt-positive cells were co-localized with gp91
phox
-positive cells (C, right, arrows) and microglia-like cells (D,
left, arrows). Although HEt-positive cells were also slightly co-localized with neurons (D, right, arrows), immunoreactivity was lower than for
gp91
phox
-positive cells and microglia-like cells. Cells were counter-stained with DAPI to show nuclei (blue). Scale bars = 20 μm (C, D).
Dohi et al. Journal of Neuroinflammation 2010, 7:41
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Page 7 of 11
Page 7
and reduced secondary damage as detected by TUNEL
staining. Moreover, we ha ve shown that the gene-defi-
cient mice have lower levels of ROS at the injury site
and widespread oxidative damage after TBI. Finally, we
demonstrate in a BV-2 microglial cell line that gp91
phox
and/or NADPH oxidase are increased in classically acti-
vated microglial cells which are activated by IFNg.
Gp91
phox
is expressed constitutively in neurons but
not in glial cells, and O
2
-
production might play a role
in neuronal homeostasis [22]. However, in neuropatho-
logical conditions including neurodegenerative diseases
and stroke, the gp91
phox
-containing NADPH oxidase has
been observed in glial cells, neurons, fibroblasts and vas-
cular endot helial cells, and seems to be involved in ROS
formation [22-29]. Microglial cell gp91
phox
appears to be
involved in the induction of neuronal damage in Parkin-
sons disease, Alzhei mers disea se, and ischemic stroke
[9,14,30,31]. In the present study, we demonstrated that
gp91
phox
is mainly expressed in microglia, and at lower
levels in neurons and astrocytes. These micro glia exhibit
features of being classically activated. We then demon-
strated that the microglial phenotypes e xpressed both
gp91
phox
and p22
phox
, which pro bably reflects the induc-
tion of NADPH oxidase in the BV-2 mouse microglial
cell line that we used. Our results reveal that NADPH
oxidase is induced in INFg-stimulated, classically acti-
vated microglial cells characterized by increased NO,
iNOS, and TNFa.
In gp91
phox-/-
mice, in situ generation of O
2
-
and an
ONOO
-
metab olite, 3-NT, were suppressed in microglia
after TBI. ONOO
-
produced by O
2
-
and NO afte r TBI
[32] induces oxidative damage, secondary brain damage
and neuroinflammation afterTBI[5,33].Wehaveveri-
fied that TUNEL-positive apoptotic-like cells in the
peri- contusional area and contusion area are suppressed
in gp91
phox-/-
mice. These results sugge st that gp91
phox
in classically activated microglia-like cell s has a harmful
role in primary and secondary brain damage after TBI.
The question of whether microglial cells play harmful
or beneficial roles in CNS injures has been widely
debated and reviewed over several decades [34-36]. The
roles of activated microglia in neuroinflammation are
thought to be complex. Class ical activation is induced
by IFNg and is related to the production of proinflam-
matory mediator s in the innate immune response.
Another form of activation, called alternative activa-
tion, is induced by IL-4 and IL-13 and, compared to
classical activation, does not result in high levels of
expression of proinflammatory mediators such as cyto-
kines and NO. The roles of alternatively activated
microglia during inflammatory process a re thought to
involve tissue repair, the production of a nti-inflamma-
tory cytokines, fibrosis, and e xtracellular matrix recon-
struction. Recently, Ohtaki et al. reported that the
injection of human mesenchymal stromal cells protects
against ischemic brain injury by modulating inflamma-
tory and immune responses th rough the alternative acti-
vation of microglia and/or mac rophages [37]. These
studies and our data suggest that controlling microglial
activation and un derstanding its mechanism and func-
tional significance follow ing TBI may open exciting new
therapeutic avenues.
The s uppression of free radica l generation and the
scavenging of free radicals after brain damage are
Figure 5 Production and ce ll identification of perox ynitrite (ONOO
-
) in wild type (Wt) (upper) and gp91
phox-/-
(lower) mice 48 hours
after TBI. ONOO
-
in Wt mice was produced in microglia-like cells (left, arrows), astrocytes (middle, arrows), and degenerated neurons (right,
arrows). In gp91
phox-/-
mice, ONOO
-
production was strongly suppressed. In neurons, only weak production of ONOO
-
was observed, but
degeneration of neurons was not seen (right, arrows). Scale bars = 20 μm.
Dohi et al. Journal of Neuroinflammation 2010, 7:41
http://www.jneuroinflammation.com/content/7/1/41
Page 8 of 11
Page 8
important therapies. Animal experiments have
supported the notion that free radical scavengers and
antioxidants dramatically reduce TBI [1,6,38]. Excessive
O
2
-
may produce destructive hydroxyl radicals (OH
-
)
and alkoxyl radicals (OR
-
) by t he iron-catalyzed Haber-
Weiss reaction. The brain is especially prone to radical
damage because it is highly enriched in easily peroxidiz-
able unsaturated fatty acid side chains and i ron. Many
studies investig ating ischemic injury suggest that inhibi-
tion of NADPH oxidase or gp91
phox
is an important
therapeutic target for neuroprotection [39,40]. Some
recent studies have demonstrated that expression of
Figure 6 Expression of gp91
phox
and p22
phox
in IFNg-exposed classically activated mouse microglial BV-2 cells. BV-2 cells were exposed
to vehicle (vehi), IFNg, IL-4, or IL-10. The levels of microglial phenotype markers (NOx, TNF a , arginase activity, iNOS, and Ym1), gp91
phox
, and
p22
phox
were evaluated in medium or cell homogenates 24 hours later. Production of (A) NOx and (B) TNFa were determined in the media. (C)
Expression of iNOS, Ym1, gp91
phox
, and p22
phox
was determined by immunoblotting with reduced samples. ß-Actin levels were used as an
internal control. IFNg-exposed BV-2 cells had increased NOx, TNFa, and iNOS levels, all of which are classical activating markers of the microglial
phenotype. In contrast, IL-4-exposed BV-2 cells had increased arginase activity and Ym1 levels, which are alternative markers of activation.
Gp91
phox
and p22
phox
levels were increased only in IFNg-exposed BV-2 cells, suggesting induction by classically activated microglial cells.
Dohi et al. Journal of Neuroinflammation 2010, 7:41
http://www.jneuroinflammation.com/content/7/1/41
Page 9 of 11
Page 9
gp91
phox
increases in brain after intracerebral hemor-
rhage, resulting in enhanced lipid peroxidation [24,41].
These studies also reported that hemorrhage volume,
brain edema, and neurological function are reduced in
gp91
phox-/-
mice, while Lo et al. reported that neurologi-
cal outcomes are improved in gp91
phox-/-
mice [42]. On
the other hand, Liu et al. reported that there are no sig-
nificant differences in mortality rate, brain water content
and intensity of oxidative stress between gp91
phox-/-
and
wild type mice in a m ouse model of subarachnoid
hemorrhage (SAH) [43]. In the present study, we have
shown that, in gp91
phox-/-
mice, gp91
phox
expressed in
classically-activated microglial-like cells plays a key role
in O
2
-
production after TBI, and that gp91
phox
-derived
O
2
-
is a key signal for contusion and cell death after
TBI. Our findings indica te that g p91
phox
inhibition and
control of microglial classically-activation might provide
a new therapeutic option by suppressing ROS genera-
tion after TBI. However, the indirect influence with TBI
by which ROS production by other NOX families and
immunecellssuchasendothelialcellorleukocytesis
currently unclear and will be an important question for
future TBI studies.
Conclusions
In conclusion, we have shown that gp91
phox
is expressed
in classical activated microglial-like cells mainly in the
peri-contusional area after TBI. An important generator
of O
2
-
is gp91
phox
during the acute phase of TBI. As
ROS derived from gp91
phox
play an important role in
primary and secondary brain damage after TBI, modula-
tion of gp91
phox
in classically activated microglia and
gp91
phox
-derived ROS may provide a new therapeutic
strategy in combating post-traumatic brain injury.
Acknowledgements
The authors thank Prof. Mary C. Dinauer for the supply the animals. The
project was supported by a Showa University Grant-in-Aid for Innovati ve
Collaborative Research Projects, by a Special Research Grant-in-Aid for the
development of Characteristic education, and a Grant-in-Aid for Scientific
Research (C) (No. 20592128, 2008) from the Japanese Ministry of Education,
Culture, Sports, Science and Technology (KD). This work was also supporte d
in part by Research on Health Sciences focusing on Drug Innovation from
The Japan Health Sciences Foundation (SS).
Author details
1
Department of Emergency and Critical Care Medicine, Showa University
School of Medicine, Shinagawa-Ku, Tokyo 142-8555, Japan.
2
Department of
Anatomy, Showa University School of Medicine, Shinagawa-Ku, Tokyo 142-
8555, Japan.
3
Department of Infectious Diseases, National Research Institute
for Child Health and Development, Setagaya-ku, Tokyo, 157-8535, Japan.
Authors contributions
KD performed the majority of experiments and data analysis, and wrote the
initial version of the manuscript. HO was involved in evaluation of microglia
using BV-2 cell. TN, SY, DS and KM were substantial contributions to western
blotting assay and immunohistochemistry. ST provided gp91 knockout mice.
KS, SS and TA supervised all experimental procedures. All of the authors
have read and approved the final version of the manuscripts.
Competing interests
The authors declare that they have no competing interests.
Received: 4 June 2010 Accepted: 26 July 2010 Published: 26 July 2010
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doi:10.1186/1742-2094-7-41
Cite this article as: Dohi et al.: Gp91
phox
(NOX2) in classically activated
microglia exacerbates traumatic brain injury. Journal of
Neuroinflammation 2010 7:41.
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  • Source
    • "Due to the important role of Nox2 in oxidative stress during diabetes and I/R, we speculate that Nox2 inhibition is the main mechanism by which dexmedetomidine exerts protection from oxidative stress during I/R in diabetic rats. In brain tissue, microglia are primary cells expressing Nox2 [39]. Western blot analysis of Iba1 (Cyto) expression indicated that activated microglia were likely to be the main resource of the increased Nox2, therefore, the effects of dexmedetomidine on Nox2 may be related to inhibition of microglia. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Transient global cerebral ischemia/reperfusion (I/R) is a major perioperative complication, and diabetes increases the response of oxidative stress and inflammation induced by I/R. The objective of this study was to determine the protective effect of dexmedetomidine against transient global cerebral ischemia/reperfusion induced oxidative stress and inflammation in diabetic rats. Methods: Sixty-four rats were assigned into four experimental groups: normoglycemia, normoglycemia + dexmedetomidine, hyperglycemia, and hyperglycemia + dexmedetomidine and all subsequent neurological examinations were evaluated by a blinded observer. Damage to the brain was histologically assessed using the TUNEL staining method while western blotting was used to investigate changes in the expression levels of apoptosis-related proteins as well as the microglia marker, ionized calcium-binding adapter molecule 1 (Iba1). Water content in the brain was also analyzed. In addition, hippocampal concentrations of malondialdehyde (MDA) and Nox2 (a member of the Nox family of NADPH oxidases), and the activity of superoxide dismutase and catalase were analyzed. Finally, changes in serum concentrations of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were detected. Results: Results showed that diabetes increased brain water content, the number of apoptotic neurons, early neurological deficit scores, oxidative stress (MDA and Nox2) and inflammation (pro-inflammatory cytokines including TNF-α and IL-6) levels following transient global I/R injury, but that these symptoms were attenuated following administration of dexmedetomidine. Conclusions: These findings suggest that dexmedetomidine can significantly alleviate damage resulting from I/R, and this mechanism may be related to a reduction in both oxidative stress and inflammation which is normally associated with I/R.
    Full-text · Article · Mar 2016 · PLoS ONE
  • Source
    • "Consistently with this, Mander and Brown [47] reported that activation of NOX or iNOS alone was relatively harmless, but their simultaneous activation was lethal because it spurred ONOO − production. Inhibition of either iNOS [48] or NOX2 [49] significantly reduced ONOO − production in CNS disease models. Collectively , we can infer that NOX2-derived O 2 − and iNOSderived NO may contribute to ONOO − formation upon microglia activation after ICH. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in intracerebral hemorrhage (ICH)-induced inflammatory injury, and the purinergic 2X7 receptor (P2X7R) is upstream of NLRP3 activation. This study aimed to investigate how P2X7R functions in ICH-induced inflammatory injury and how the receptor interacts with the NLRP3 inflammasome. Methods: Rats were treated with P2X7R small interfering RNA (siRNA) 24 h before undergoing collagenase-induced ICH. A selective P2X7R inhibitor (blue brilliant G, BBG) or a peroxynitrite (ONOO(-)) decomposition catalyst (5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) [FeTPPS]) was injected 30 min after ICH. Brain water content, hemorrhagic lesion volume, and neurological deficits were evaluated, and western blot, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were carried out. Results: Striatal P2X7R and NLRP3 inflammasomes were activated after ICH. Gene silencing of P2X7R suppressed NLRP3 inflammasome activation and interleukin (IL)-1β/IL-18 release and significantly ameliorated brain edema and neurological deficits. Additionally, enhanced NADPH oxidase 2 (NOX2, gp91(phox)) and inducible nitric oxide synthase (iNOS), as well as their cytotoxic product (ONOO(-)) were markedly attenuated by BBG treatment following ICH. This was accompanied by downregulations of the inflammasome components, IL-1β/IL-18 and myeloperoxidase (MPO, a neutrophil marker). Most importantly, inflammasome activation and IL-1β/IL-18 release were significantly inhibited by ONOO(-) decomposition with FeTPPS. Conclusions: Our findings implicate that P2X7R exacerbated inflammatory progression and brain damage in ICH rats possibly via NLRP3 inflammasome-dependent IL-1β/IL-18 release and neutrophil infiltration. ONOO(-), a potential downstream signaling molecule of P2X7R, may play a critical role in triggering NLRP3 inflammasome activation.
    Full-text · Article · Oct 2015 · Journal of Neuroinflammation
  • Source
    • "Activated microglia, however , could significantly induce production of a large array of inflammatory cytokines, like interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α) [12] and inducible nitric oxide synthase (iNOS) [13], leading to the neuronal cell damage of inflammatory surroundings. The bystander effect of activated macroglia plays a decisive role in the pathogenesis of traumatic brain injury (TBI), intracerebral hemorrhage (ICH), manganese poisoning, and Lyme neuroborreliosis (LNB)14151617. Without the involvement of lipopolysaccharide (LPS), T. gondii glycosylphosp-hatidylinositols (GPIs), which are known to activate TLR2 and TLR4 receptors, may trigger production of inflammatory cytokines by mouse macrophages [18] . "
    [Show abstract] [Hide abstract] ABSTRACT: Background A plethora of evidence shows that activated microglia play a critical role in the pathogenesis of the central nervous system (CNS). Toxoplasmic encephalitis (TE) frequently occurs in HIV/AIDS patients. However, knowledge remains limited on the contributions of activated microglia to the pathogenesis of TE. Methods A murine model of reactivated encephalitis was generated in a latent infection with Toxoplasma gondii induced by cyclophosphamide. The neuronal apoptosis in the CNS and the profile of pro-inflammatory cytokines were assayed in both in vitro and in vivo experiments. Results Microglial cells were found to be activated in the cortex and hippocampus in the brain tissues of mice. The in vivo expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) were up-regulated in TE mice, and accordingly, the neuronal apoptosis was significantly increased. The results were positively correlated with those of the in vitro experiments. Additionally,apoptosis of the mouse neuroblastoma type Neuro2a (N2a) remarkably increased when the N2a was co-cultured in transwell with microglial cells and Toxoplasma tachyzoites. Both in vivo and in vitro experiments showed that minocycline (a microglia inhibitor) treatment notably reduced microglial activation and neuronal apoptosis. Conclusions Activated microglia contribute to neuronal apoptosis in TE and inhibition of microglia activation might represent a novel therapeutic strategy of TE.
    Full-text · Article · Aug 2014 · Parasites & Vectors
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