Background
Traumatic brain injury (TBI) is a serious condition in emergency medicine, and its pathophysiological profile is varied and complicated. One of the neurotoxic factors thought to be involved is oxidative stress [
1,
2]. A large number of studies have reported that oxidative stress, which generates reactive oxygen species (ROS), plays a key role in the development of TBI [
1,
3,
4]. Consequently, one of the most obvious ways to manage TBI may be to control ROS generation [
1] given 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 important free radical, and is the source of other ROS that lead to lipid 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 homologs of the catalytic subunit of the enzyme, gp91
phox, also termed NOX2, 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 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 founder, and experiments were performed in age- and weight-matched animals.
Controlled cortical impact model
Mice were anesthetized with 2% sevoflurane in 70% N
2O and 30% O
2. A controlled cortical impact was made using a pneumatically 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 mouse BV-2 cell line was obtained from Interlab Cell Line Collection (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-glutamine [all from GIBCO/BRL, Grand Island, NY]). The cells were grown at 37°C in a humidified 5% CO
2 incubator. After harvesting, the cells were washed with PBS twice and resuspended with experimental medium (Dulbecco's modified Eagle's 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 IFNγ (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 centrifugation. 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 contralateral hemispheres. 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 centrifuged at 12,000 × g for 10 minutes on ice. BV-2 samples were sonicated for 10 seconds with lysis buffer to prepare cell suspensions.
After determination of the protein concentration (BCA protein assay, Thermo Fisher Scientific, Waltham, MA), appropriate amounts of samples were electrophoresed. The separated proteins were then transferred to polyvinylidinene fluoride membranes (Bio-Rad, Hercules, CA). After blocking with 2% Blockace (DS Pharma, Osaka, Japan), the membranes were probed with primary antibodies. After washing, the membrane was probed with horseradish peroxidase (HRP)-conjugated secondary antibodies. The protein bands were detected by chemiluminescence (SuperSignal West Dura Extended Duration Substrate; Pierce, Rockford, 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
2O
2 and then incubated with PBS containing 5% normal horse serum to mask nonspecific reactions. Next, the sections were incubated with antibody raised against gp91
phox [
18]. One day later, the sections were rinsed and incubated with biotinylated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA), and then with an avidin-biotin complex solution (Vector Laboratories, Burlingame, CA) followed by diaminobenzidine (DAB; Vector) as a chromogen.
A similar procedure was used for multiple immunostaining, except that the sections were not incubated with 0.3% H
2O
2, and were incubated with Alexa-labeled fluorescence secondary antibodies. Primary and secondary 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 fluorescence and immunolabeling were detected using a confocal laser microscope (AX-10, Zeiss; Oberkochen, Germany).
Table 1
Antibodies used for immunoblotting (IB) and immunohistochemistry (IHC)
Primary antibody (clone #)
|
gp91phox | Human gp91 | Rabbit | See ref 18 | | 4,000 (IB) 200 (IHC) |
p22phox | 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 |
β-Actin (AC-74) | Mouse β-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 |
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 delineated by examining differences between the ipsilateral and contralateral regions in the center slice of injured brain and measured by using NIH Image software.
http://rsb.info.nih.gov/nih-image/about.html.
Evaluation of apoptosis-like cell death
To determine neural apoptosis-like cell death, terminal deoxynucleotidyl transferase-mediated dUTP end-labeling (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 ipsilateral hemisphere was then counted and compared gp91phox-/- with Wt mice in a similar cortical region (40 × magnification).
In situ detection of O2-
Production of O
2- was determined by
in situ detection of oxidized hydroethidium (HEt) [
19]. With the animal placed under anesthesia, the HEt solution was administered (1 mg/mL 0.9% NaCl with 1% DMSO) into the jugular vein (
n = 3 per group) 48 hours 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. Fluorescence was detected using confocal laser microscopy (AX-10, Zeiss, Germany)
NO and TNFα measurement in media
Levels of NO and TNFα production are markers of classically activated microglia [
20]. NO production was measured using the Griess method (Dojindo, Kumamoto, Japan) as total NO (NO
2- and NO
3-). TNFα 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 alternatively activated microglia [
20]. Arginase activity was measured according to a previous paper [
21] with minor modification. In brief, the cell homogenate was mixed with equal volumes of prewarmed 50 mM Tris-HCl, pH 7.5 containing 10 mM MnCl
2 and incubated for 15 minutes at 55°C. The mixture was incubated in 0.25 M
L-arginine for 60 minutes at 37°C to produce urea from arginine and the reactions were stopped by adding Stop solution (H
2SO
4/H
3PO
4/H
2O, 1:3:7). Then, 1% (final concentration) 1-phenyl-1, 2-propanedione-2-oxime (ISPF) in ethanol was added to the solution, which was heated at 100°C for 45 min. The reaction between urea and ISPF produced a pink color, and absorption was measured at 540 nm.
Statistical analysis
Data are expressed as mean ± SE for in vivo experiments. Data are expressed as mean ± SD for in vitro experiments Statistical comparisons were performed using the Student's t tests and two-way analysis of variance (ANOVA) as appropriate. P values less than 0.05 were considered statistically significant.
Discussions
We demonstrate here that gp91phox is increased in the ipsilateral hemisphere after TBI and specifically in amoeboid-shaped microglial cells. Mice that are gene-deficient for gp91phox exhibit reduced primary cortical damage, as evidenced by reduced areas of contusion, and reduced secondary damage as detected by TUNEL staining. Moreover, we have shown that the gene-deficient 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 gp91phox and/or NADPH oxidase are increased in classically activated microglial cells which are activated by IFNγ.
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 neuropathological conditions including neurodegenerative diseases and stroke, the gp91
phox-containing NADPH oxidase has been observed in glial cells, neurons, fibroblasts and vascular endothelial 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 Parkinson's disease, Alzheimer's disease, 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 microglia exhibit features of being classically activated. We then demonstrated that the microglial phenotypes expressed both gp91
phox and p22
phox, which probably reflects the induction of NADPH oxidase in the BV-2 mouse microglial cell line that we used. Our results reveal that NADPH oxidase is induced in INFγ-stimulated, classically activated microglial cells characterized by increased NO, iNOS, and TNFα.
In gp91
phox-/- mice,
in situ generation of O
2-and an ONOO
- metabolite, 3-NT, were suppressed in microglia after TBI. ONOO
- produced by O
2- and NO after TBI [
32] induces oxidative damage, secondary brain damage and neuroinflammation after TBI [
5,
33]. We have verified that TUNEL-positive apoptotic-like cells in the peri-contusional area and contusion area are suppressed in gp91
phox-/- mice. These results suggest that gp91
phox in classically activated microglia-like cells 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. Classical activation is induced by IFNγ and is related to the production of proinflammatory mediators in the innate immune response. Another form of activation, called "alternative activation," 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 cytokines and NO. The roles of alternatively activated microglia during inflammatory process are thought to involve tissue repair, the production of anti-inflammatory cytokines, fibrosis, and extracellular matrix reconstruction. Recently, Ohtaki et al. reported that the injection of human mesenchymal stromal cells protects against ischemic brain injury by modulating inflammatory and immune responses through the alternative activation of microglia and/or macrophages [
37]. These studies and our data suggest that controlling microglial activation and understanding its mechanism and functional significance following TBI may open exciting new therapeutic avenues.
The suppression of free radical generation and the scavenging of free radicals after brain damage are 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 the iron-catalyzed Haber-Weiss reaction. The brain is especially prone to radical damage because it is highly enriched in easily peroxidizable unsaturated fatty acid side chains and iron. Many studies investigating ischemic injury suggest that inhibition of NADPH oxidase or gp91
phox is an important therapeutic target for neuroprotection [
39,
40]. Some recent studies have demonstrated that expression of gp91
phox increases in brain after intracerebral hemorrhage, 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 neurological outcomes are improved in gp91
phox-/- mice [
42]. On the other hand, Liu et al. reported that there are no significant differences in mortality rate, brain water content and intensity of oxidative stress between gp91
phox-/- and wild type mice in a mouse 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 indicate that gp91
phox inhibition and control of microglial classically-activation might provide a new therapeutic option by suppressing ROS generation after TBI. However, the indirect influence with TBI by which ROS production by other NOX families and immune cells such as endothelial cell or leukocytes is currently unclear and will be an important question for future TBI studies.
Competing interests
The authors declare that they have no competing interests.
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.