Dev Neurosci 2013;35:241–254 DOI: 10.1159/000346159
Phenotype and Secretory Responses to Oxidative Stress in Microglia
Praneeti Pathipati a Sebastian Müller c Xiangning Jiang a Donna Ferriero a, b
Departments of a Neurology and b Pediatrics, University of California San Francisco, San Francisco, Calif. , USA; c Medical Department, Salem Medical Center and Alcohol Research Center, University of Heidelberg, Heidelberg , Germany
dized and reduced) content and fixed cells were labeled for M1 and M2a phenotype markers. Overall, it is evident that microglial exposure to continuous H 2 O 2 has pleiotropic and biphasic effects. Continuous exposure to very low levels of H 2 O 2 is more damaging to cell survival than higher bolus doses at 18 h, and can produce considerably high levels of pro- and anti-inflammatory cytokines by 18 h. Significantly high levels of various chemokines/chemotactic molecules such as G-CSF, MIP-1b and MIP-2 are also produced in re-sponse to continuous low-dose H 2 O 2 by 18 h. Interestingly, no prominent cytokine responses were seen with bolus treatment at any of the time points studied. H 2 O 2 exposure promotes an M2a microglial phenotype in the absence of IL-4/IL-13 signaling, suggesting a wound-healing role for mi-croglia and a delayed activation mechanism for H 2 O 2 after such an insult. Together, these specific effects can be used to clarify the microglial cell responses following injury in the immature brain.
Microglia are the resident immune cells of the brain and play an important role in initiating and mediating neuroinflammation. Upon activation, they produce a
Key Words
Hydrogen peroxide · Reactive oxygen species · Nitric oxide · Antioxidants · Neuroinflammation · Development · Inflammatory mediators · Cytokines
Abstract
The neonatal brain is particularly susceptible to oxidative stress. Our group has previously shown that following hy-poxic-ischemic injury, hydrogen peroxide (H 2 O 2 ) levels rise significantly particularly in the neonatal brain and are sus-tained for up to 7 days. This rapidly accumulated H 2 O 2 is det-rimental in the iron-rich immature brain as it can lead to the generation of dangerous free radicals that can cause exten-sive injury. To date, there is limited literature on the effects of increased H 2 O 2 levels on microglial cells, which have been extensively implicated in the ensuing inflammatory injury. Microglial cultures were derived from the P1 mouse brain and exposed to either bolus concentrations of H 2 O 2 (15 or 50 μ M ) or varying concentrations of continuous exposure for 4, 18 or 24 h. Continuous exposure of microglia to H 2 O 2 was generated using the glucose oxidase-catalase system gener-ating levels of H 2 O 2 <10 μ M . Reactive oxygen species and nitric oxide expression were measured. Conditioned medi-um was collected and analyzed for secreted cytokine levels. Treated cell extracts were processed for glutathione (oxi-
Received: August 21, 2012 Accepted after revision: November 26, 2012 Published online: March 16, 2013
Praneeti Pathipati UCSF Mission Bay Campus 675 Nelson Rising Lane, Rm 494 San Francisco, CA 94158 (USA) E-Mail pathipatip @ neuropeds.ucsf.edu
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variety of proinflammatory mediators such as tumor ne-crosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, monocyte chemotactic protein 1 (MCP-1, CCL2), nitric oxide (NO) and various reactive oxygen species (ROS) [1, 2] which initiate bystander injury. However, after the initial ‘toxic’ phase, these functionally plastic cells may also mediate important anti-inflammatory and regener-ative functions for the resolution of tissue injury [3] . It is possible that the intensity of the injury/insult actually determines if microglial function is beneficial or detri-mental [4] . Since neuroinflammation is a characteristic of several disease states in the brain, it is important to clarify the role of microglia in various states.
Hypoxic-ischemic (HI) injury to the brain is a signifi-cant cause of mortality and severe neurologic disability. In the developing immature brain, which is highly depen-dent on sustained blood flow for energy and nutrients, it is highly damaging and as a result, a common cause of long-term disability in newborns [5] . HI injury triggers a cascade of neurotoxic events involving energy failure, glutamate release, activation of N-methyl- D -aspartate re-ceptors, influx of calcium and formation of NO, all of which lead to mitochondrial dysfunction and energy fail-ure [6] . While under normal conditions several enzymat-ic and nonenzymatic antioxidants provide protection from the deleterious effects of the resulting ROS, in the face of energy failure these defenses fail. Furthermore, the levels of antioxidants in the immature brain are lower and when coupled with the abundant availability of ‘free’ re-dox-reactive iron, there is selective vulnerability of the immature brain to oxidative damage [7] . Since there is significant accumulation of hydrogen peroxide (H 2 O 2 ) in particular, coupled with lower glutathione peroxidase levels [8, 9] , H 2 O 2 -mediated injury processes can occur in the developing brain.
Several studies suggest that microglial activation is det-rimental to the immature brain [10–12] . On the contrary, however, there is evidence that neither inhibiting microg-lial activation [13, 14] nor depleting microglial numbers [15] is beneficial, suggesting that microglia can in fact be neuroprotective [15, 16] . Since microglial cells are the pri-mary responders following injury, and injury in the im-mature brain leads to a significant accumulation of H 2 O 2 [17] , it is important to identify the effects of sustained H 2 O 2 levels on microglia. It is hypothesized that while at low concentrations H 2 O 2 can function as a (possibly harmless) signaling molecule, at higher concentrations, H 2 O 2 will have a detrimental effect on microglial cells.
One of the major caveats of utilizing bolus treatments of H 2 O 2 however, is that bolus treatments expose cells to
artificially high levels of H 2 O 2 as compared to more physiological levels (less than 10 μ M ) of H 2 O 2 that are released by inflammatory cells [18] . Furthermore, H 2 O 2 boli have a very short half-life under cell culture condi-tions [19, 20] . In order to overcome these drawbacks, we tested our hypothesis using an enzymatic system (glu-cose oxidase-catalase; GOX-CAT) that can continuously generate and sustain H 2 O 2 for up to 24 h at lower, more physiological doses of less than 10 μ M [21] . Primary mu-rine microglial cells were used in this study and compar-isons of continuous exposure were made to bolus treat-ments in order to delineate the specific effects of sus-tained H 2 O 2 exposure. We hypothesize that at lower dos-es such as at the ×5,000 CAT dose and 15/50 μ M bolus dose, H 2 O 2 exposure could serve as a preconditioning mechanism [22] that would ‘prime’ microglia for future insults.
Materials and Methods
Materials The following reagents were all sourced from the Cell Culture
Facility at the University of California San Francisco: Hanks’ bal-anced salt solution, Dulbecco’s modified Eagle’s medium (DMEM; phenol red-free, 4.5 g/l glucose, with no pyruvate), penicillin-streptomycin (P/S), fetal bovine serum (FBS; Axenia Biologix) and 0.05% trypsin. Glucose oxidase (G0543), catalase (C3155), H 2 O 2 (H1009), H 2 DCFDA (#D6883), sulfosalicylic acid (S2130), DTNB (D8130), β-NADPH (N7505), glutathione reductase (G3664), glu-tathione (GSH; G4251), glutathione disulfide (GSSG; G6654), and 2-vinylpyridine (132292) were from Sigma (St. Louis, Mo., USA). Neurobasal medium (12348-017), B27 (17504044), B27 antioxi-dants (10889038), GlutaMax I (35050), poly- D -lysine (P7280), Griess reagent (G7921), Live-Dead Assay (L3224) and secondary antibodies [goat anti-mouse Alexa 488, goat anti-rabbit Alexa 568 (1: 200, 11,001 and 11,011, respectively), donkey anti-goat Alexa 488 (1: 400, 11,055)] were from Invitrogen (Carlsbad, Calif., USA). Lipopolysaccharide (LPS strain 055:B55, 203) was from LIST Bio-chemicals (Campbell, Calif., USA). Iba-1 (019-19741), glial fibril-lary acid protein (3655), O4 (MAB345) and neuron-specific eno-lase (AO587) were from WAKO chemicals (Japan), Cell Signalling (Danvers, Mass., USA), Millipore (Billerica, Mass., USA) and DAKO (Denmark), respectively. LDH (11644793001) was from Roche. Primary antibodies used for microglial activation marker staining were mouse anti-human arginase 1 (1: 50, 610708), rabbit anti-mouse iNOS (1: 50, 610332) and mouse anti-rat CD32 (1: 50, 550270) from BD Biosciences (Franklin Lakes, N.J., USA) and rab-bit anti-human CD206 (1: 100, sc48758) from Santa Cruz Biotech-nology (Santa Cruz, Calif., USA).
Microglial Culture and H 2 O 2 Treatment Microglial Culture Animals were sacrificed in accordance with guidelines from the
Institutional Animal Care and Use Committee at the University of California San Francisco. Mixed glial cultures were prepared from
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P1 C57BL/6 mice (Charles River Laboratories Inc., Wilmington, Mass., USA). Brains were isolated, stripped of meninges, mechan-ically dissociated and trypsinized for 15 min at 37 ° C. Following resuspension in DMEM supplemented with 1% P/S and 10% FBS, cells were seeded (2 brains/T-75 flask) and incubated at 37 ° C/5% CO 2 . Medium was changed the next day and flasks left untouched for 10 days. After 10–14 days, flasks were shaken for 1–2 h at 200 rpm, 37 ° C to lift off microglial cells which were then plated at a density of 1.25 × 10 5 /cm 2 into 24-well (for viability, mRNA, ROS expression and immunofluorescence studies) or 12-well (protein/cell extracts) plates in DMEM supplemented with 1% P/S and 2% FBS overnight and used the next day. Cells were plated on cover-slips (13 mm for 24-well plates) where utilized for immunofluores-cence. Astroglial contamination was confirmed to be less than 5% using immunofluorescence (Iba-1 for microglia, glial fibrillary acid protein for astrocyte and O4 for oligodendrocyte labeling; data not shown).
Microglia Stimulation with H 2 O 2 Two methods of stimulation were used – continuous using the
GOX-CAT system and bolus treatment using exogenously applied H 2 O 2 .
The GOX-CAT method is an enzymatic method that can be used to generate low, continuous H 2 O 2 for over 24 h [21] . GOX is a stable enzyme that remains fully active over 24 h at 37 ° C and generates H 2 O 2 by consuming oxygen while CAT degrades H 2 O 2 back to water and oxygen and is not saturable even at molar con-centrations of H 2 O 2 . This means that while GOX generates H 2 O 2 over 24 h, CAT continuously breaks it down in a typical exponen-tial kinetic fashion that is directly dependent on the H 2 O 2 concen-tration. Accordingly, stable H 2 O 2 concentrations are generated that only depend on the ratio of enzyme activities [23] . Thus, by regulating the specific dilutions of CAT (and thus the rate of H 2 O 2 breakdown) in the medium, it is possible to produce controlled amounts of H 2 O 2 continuously for at least 24 h within a variability of 20% [21] . Under equilibrium conditions, H 2 O 2 steady-state concentrations can be directly calculated as the ratio of K GOX /K CAT [21] . Appropriate conditions were used to avoid hypoxia with re-gard to GOX activity and medium volume, since a high GOX ac-tivity and a large medium volume could be used to independently induce hypoxia [21, 23] . No significant hypoxia is induced for 12- and 24-well plates using a GOX activity of 1: 100,000 and medium volumes of 1 and 0.5 ml, respectively [21] .
In all experiments here, GOX was kept constant at 1: 100,000 while CAT dilution was varied (1: 5,000, 1: 20,000, 1: 80,000) result-ing in H 2 O 2 levels of around 1–3 μ M . In addition, two standard bolus conditions were used for H 2 O 2 at 15 and 50 μ M . Since serum can contain CAT, all H 2 O 2 (continuous and bolus) treatments were carried out in serum-free DMEM with 1% P/S. LPS was used as a positive control at a concentration of 100 ng/ml and was di-luted in 2% FBS DMEM [24] . Serum-free DMEM served as nega-tive control. Following overnight plating in 2% FBS DMEM, mi-croglial medium in the plates was replaced with prewarmed test medium (containing GOX-CAT/bolus H 2 O 2 /LPS or negative con-trol) for 4, 18 or 24 h. All assays were run in triplicate and repeated independently at least 3 times.
Viability Assay Microglial viability in response to H 2 O 2 treatment was assessed
using the LDH assay according to manufacturer’s instructions.
LDH release into the medium was compared to total LDH in the medium following cell lysis.
Nitrite Assay Nitrite levels in medium were used as a surrogate measure of
NO using the Griess reagent according to manufacturer’s proto-cols. Briefly, at the end of each time point, 150 μl of medium from each well was mixed with 20 μl of Griess reagent and 130 μl of de-ionized water for 30 min at room temperature. Absorbance was measured and expressed relative to the reference (sodium nitrite) sample at 548 n M .
ROS Assay Cellular ROS content was measured using the H 2 DCFDA re-
agent. At the end of each time point, cells were loaded with 20 μ M H 2 DCFDA for 2 h at 37 ° C. Following a wash with prewarmed Hanks’ balanced salt solution, fluorescence absorption was mea-sured at 485 Ex /535 Em nm.
Mitochondrial Superoxide Assay Mitochondrial superoxide levels were measured using the Mi-
toSox Red indicator dye. As it is readily oxidized by superoxide but not by other ROS- or reactive nitrogen species-generating systems, it would give an accurate measure of superoxide levels in response to H 2 O 2 treatment. At the end of each time point, cells were load-ed with 5 μM MitoSox dye in prewarmed serum-free DMEM for 10 min at 37 ° C. Following 2 washes in the same medium, fluores-cence absorption was measured at 485 Ex /590 Em nm.
Antioxidant (GSH/GSSG) Levels GSH/GSSG levels were measured using a modified Tietze recy-
cling assay as described in Rahman et al. [25] . Briefly, cell extracts were prepared using an extraction buffer containing 0.1% Triton X-100 and 0.6% sulfosalicylic acid, sonicated for 2–3 min and sub-jected to a –80 ° C freeze-thaw cycle to ensure complete cell lysis. The supernatants were then used in a kinetic assay in a 96-well microtiter plate using DTNB, glutathione reductase and β-NADPH. Absorbance was read at 412 nm and measurements were taken ev-ery 50 s for 4 min. The rate of change in absorbance per minute was calculated to give the rate of 2-nitro-5-thiobenzoic acid forma-tion and actual total GSH (GSH + 2GSSG) in samples using linear regression to calculate values obtained from the standard curve. For GSSG, the same procedure was repeated but on samples pre-treated for 1 h with 2-vinylpyridine [which covalently reacts with GSH (but not GSSG) and thus prevents the rapid oxidation of GSH to GSSG] and triethanolamine.
Cytokine Measurements Multiplex Assay. Cytokine protein expression in monocyte-
conditioned medium was assessed according to the manufactur-er’s instructions on a mouse 25-plex cytokine microbead assay kit which allows for simultaneous detection of 25 inflammatory mol-ecules in a single 75-μl sample including G-CSF, GM-CSF, IFN-γ, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IP-10, KC-like, MCP-1, MIP-1α, RANTES and TNF-α. Results were analyzed using a Bio-plex workstation (Bio-Rad) and levels were normalized to the total amount of pro-tein in the appropriate well. Protein concentrations were deter-mined using the BioRad DC protein assay. Data are expressed as picograms per microgram of protein. The level of sensitivity for
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each microbead cytokine standard curve ranged from 1 to 35 pg/ml. Two 96-well assays were performed in duplicate with each plate containing monocyte-conditioned medium from 2 indepen-dent experiments from each of the 3 time points.
Immunofluorescence for Microglial Marker Staining Cells were fixed in 4% paraformaldehyde in 0.1 M phosphate
buffered saline (PBS) for 15 min. Following 3 washes in PBS, the coverslips with adherent cells were used for double immunofluo-rescence labeling. Following a 1-hour block at room temperature in 10% normal goat or donkey serum diluted in PBS with 8 mg/ml bovine serum albumin and 0.1% Triton, they were incubated with primary antibodies against iNOS, Arginase 1, CD32 and CD206 diluted in 2% blocking serum overnight at 4 ° C. The next day after 3 washes in PBS with 0.1% Triton, cells were incubated in secondary antibodies for 2 h at room temperature, washed and coverslips mounted on glass slides using Vectashield with DAPI. Coverslips were imaged at ×10 and ×25 using a Zeiss Axiovert 100 microscope and Volocity software (Improvision, Waltham, Mass., USA).
Statistics All statistical analyses were performed with GraphPad Prism
5.0 (GraphPad Software, San Diego, Calif., USA). Two-way analy-sis of variance and Bonferroni post hoc tests were applied with replicate number and treatment as factors for each time point for all assays except the multiplex assay. For the multiplex assay, for each treatment group from each time point, the levels of inflam-matory mediators in 4 independent assays were run in duplicate and results averaged to give a mean. All results are expressed as means ± SEM and significance was accepted at p ≤ 0.05.
Results
Microglial Viability Is Dependent on the Nature of H 2 O 2 Exposure H 2 O 2 is a strong oxidant and at high levels can cause
extensive damage via production of damaging free radi-cals. In order to study the effects of increasing concentra-tions of H 2 O 2 , microglial cells were exposed to dilutions of CAT while GOX was kept constant. As generation of H 2 O 2 by GOX is almost independent of oxygen and glu-cose in high-glucose media [21, 26] , varying the CAT concentration exclusively controls the concentration of H 2 O 2 in culture at any time. Figure 1 a shows the dose re-sponse to increasing CAT dilution (i.e. increasing H 2 O 2 concentration) at 6 and 24 h. As the CAT concentration decreases, there is a gradual increase in the amount of LDH released into the medium. Figure 1 b shows a com-parison between continuous and bolus treatments at 4, 18 and 24 h. Low, continuous H 2 O 2 (approx. 1–5 μ M with ×80,000 CAT) leads to a significantly higher LDH release at 18 and 24 h when compared to that of any of the bolus H 2 O 2 doses (p < 0.01; fig. 1 b).
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Fig. 1. Low, continuous H 2 O 2 can be more detrimental than high bolus doses. a Dose response to continuous doses of H 2 O 2 . The GOX concentration was maintained constant while the CAT con-centration was varied. As the CAT concentration increases, anincrease in cell death is evident, as measured by the LDH release
assay. When compared to effects of bolus H 2 O 2 , low continuous H 2 O 2 appears to be more detrimental than higher bolus doses. Shown are means ± SEM. * p < 0.05; * * p < 0.01; * * * p < 0.001. All significances shown are against control unless where indicated otherwise. LPS (positive control) was added at 100 ng/ml.
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Superoxide Anions Contribute to the Early Increase in ROS Levels while Nitrite Production Is Predominant at 24 h Microglial cells produce ROS in response to injury [27,
28] . Hence nitrite and ROS levels were measured in mi-croglial cells exposed to continuous or bolus doses for 4, 18 or 24 h.
Using the Griess assay, nitrite levels were detectable in monocyte-conditioned medium only after 24 h of H 2 O 2 ex-posure (p < 0.001; fig. 2 a). Significantly elevated levels of nitrite were evident at the highest continuous dose (×80,000 CAT, p < 0.001). Interestingly, both bolus doses (15 and 50 μ M ) exhibited significantly lower nitrite levels when com-pared to control (p < 0.01 and p < 0.001, respectively) and
all continuous doses (p < 0.001 for all continuous vs. 50 μ M and ×80,000 CAT vs. 15 μ M ). Cellular ROS levels were de-termined using H 2 DCFDA [29, 30] ( fig. 2 b). Elevation in cellular ROS was seen as early as 4 h across all continuous doses tested although significantly elevated levels were seen only at 18 h (p < 0.01 for ×20,000 CAT and p < 0.05 for ×80,000 CAT), which were reduced by 24 h. On the other hand, ROS levels were significantly elevated from 4 h on-wards for both the bolus doses (overall p < 0.01 for 15 and 50 μ M ) and remained elevated even at 24 h. In order to de-termine the contribution of superoxide ions to cellular ROS levels, MitoSox Red, a selective indicator of mitochondrial superoxide [29, 31] , was employed. Mitochondrial super-oxide levels were significantly elevated by 4 h in the ×20,000
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* ************* ***** Fig. 2. Nitrite levels are elevated by 24 h while elevated levels of ROS are seen as early as 4 h, with superoxide levels contributing to a large portion of these. a Nitrite levels in the medium as measured by the Griess assay were elevated only after 24 h of treatment. Lev-els produced with ×80,000 CAT dilution are significantly higher than those produced by a 50 μ M bolus dose. b Cellular ROS levels (measured using the H 2 DCFDA indicator) are elevated as early as 4 h after initiation of H 2 O 2 exposure, especially in the bolus doses. It is likely that most of the ROS seen at 4 and 18 h are superoxide ions as is shown in c . Mitochondrial superoxide levels, however, are decreased by 24 h. Superoxide levels were measured using the MitoSox Red indicator. Shown are means ± SEM. * p < 0.05; * * p < 0.01; * * * p < 0.001. All significances shown are against con-trol unless where indicated otherwise. LPS (positive control) was added at 100 ng/ml.
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CAT and both bolus doses (p < 0.05 and p < 0.001, respec-tively), remained elevated at 18 h (p < 0.001 for all three doses) but dropped back to control levels by 24 h ( fig. 2 c).
Antioxidant Levels Are Increased with Continuous H 2 O 2 Previous studies have identified cultured microglial
cells to have a significantly high GSH content and spe-cific activity when compared to astroglia and neurons [32] . Although under normal conditions they only have marginal amounts of oxidized glutathione (GSSG), oxi-dative stress rapidly increases GSSG levels [32] . We mea-sured both oxidized and total GSH levels in our study us-ing the modified Tietze assay [25] ( fig. 3 ). GSH levels were significantly higher at 24 h compared to 4 h at the ×5,000 and ×20,000 CAT dose (p < 0.05) but lower at 18 and24 h than at 4 h at the ×80,000 CAT dose (p < 0.05; fig. 3 a). The overall p value was <0.001. The contribution of GSSG to total GSH (since total GSH = GSH + 2GSSG) appeared to be minimal at most doses ( fig. 3 b). It is only at the ×5,000 CAT dose that significantly increased levels of GSSG were evident at 18 h (p < 0.05).
Low Continuous H 2 O 2 Mimics LPS in Terms of Cytokine Expression Depending on their activation state, microglial cells se-
crete various pro- and anti-inflammatory cytokines, che-
mokines, as well as other signaling molecules. Overall, several inflammatory mediator levels were seen elevated only at the lowest dose of continuous H 2 O 2 (×5,000 CAT), with markedly higher levels at 18 and 24 h. These cyto-kines have been broadly divided into pro- and anti-in-flammatory cytokines and chemokines/chemotacticmolecules. Of the proinflammatory cytokines ( fig. 4 ),although only IL-15 at ×5,000 CAT was significantly higher than control at 18 h (p < 0.05), IL-1α, IL-1β, IL-6, IL-15, IL-17, TNF-α and IFN-γ levels were all at least 2-fold higher compared to control (at least 100 pg/mg protein for ×5,000 CAT at 18 and 24 h compared to less than 50 pg/mg for control, not significant). Other proin-flammatory cytokines that exhibited less than a 2-fold in-crease were IL-2, IL-5, IL-7, IL-12 p40 and p70 (not shown). Regarding the anti-inflammatory cytokine levels ( fig. 5 ), they were not significantly different to control at any of the doses tested, although IL-9, IL-10 and IL-13 levels were at least 2-fold higher than control at the ×5,000 CAT dose (not significant). Almost all chemokines/che-motactic molecules exhibited increased levels, with G-CSF, MIP-1b and MIP-2 showing significantly elevated levels at 18 and 24 h with the ×5,000 CAT dose (p < 0.1 to p < 0.001; fig. 6 ). All other chemokines/chemotactic mol-ecules expressed at least 2-fold higher levels than control (MCP-1, RANTES, GM-CSF and KC; fig. 6 , not signifi-cant). Overall, when compared to responses to LPS treat-
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Fig. 3. Low continuous H 2 O 2 elicits a greater change in antioxidant levels compared to bolus doses. a Total glutathione (GSH + 2GSSG) levels. b Oxidized (GSSG) levels. GSH content was mea-sured using a modified Tietze assay. Shown are means ± SEM.
* p < 0.05; * * p < 0.01; * * * p < 0.001. All significances shown are against control unless where indicated. LPS (positive control) was added at 100 ng/ml. Please note that data shown for 50 μ M are from 2 replicates.
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Fig. 4. Secretion of proinflammatory cytokines in response to H 2 O 2 . Shown are means ± SEM. * p < 0.05; * * p < 0.01; * * * p < 0.001. All significances shown are against control unless where indicated. LPS (positive control) was added at 100 ng/ml.
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ment (100 ng/ml), although H 2 O 2 -treated microglia ex-hibited a similar expression pattern, the responses were largely attenuated. It is possible that some of these ob-served changes in cytokine levels were due to differences in microglia viability as significant cell death is seen par-ticularly with the ×80,000 CAT dose (p < 0.001; fig. 1 ).
H 2 O 2 Induces an M2a Phenotype in Microglia Microglia/macrophages can become polarized to pro-
and anti-inflammatory states that exhibit either harmful or protective and reparative properties, depending on the disease context [3, 33, 34] . Here, using antibodies direct-ed against M1 (iNOS and CD32) and M2 (CD206, Argi-nase 1) activation markers, immunofluorescence identi-
fied microglial phenotype in response to H 2 O 2 exposure. Using Western blotting, protein expression of these markers was confirmed. For clarity, only iNOS and Argi-nase 1 staining is shown in figure 7 a. At 4 h, there was weak staining for both iNOS and Arginase 1 in all cells (including control LPS and all H 2 O 2 wells), with slightly higher iNOS levels than Arginase 1 ( fig. 7 a, 4 h column). Some iNOS+ cells were still visible in all H 2 O 2 -treated wells even at 18 h (not shown) although by 24 h, very few iNOS+ cells were visible in the H 2 O 2 -treated cells with Arginase 1+ cells visible all over ( fig. 7 a, 24 h column). CD206 staining was visible in H 2 O 2 -treated cells by 18 h and persisted at 24 h, while CD32 staining was weak in all wells at all time points (results not shown). This pattern
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Fig. 5. Secretion of anti-inflammatory cytokines in response to H 2 O 2 . Shown are means ± SEM. * p < 0.05; * * p < 0.01; * * * p < 0.001. All significances shown are against control unless where indicated. LPS (positive con-trol) was added at 100 ng/ml.
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0
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Fig. 6. Secretion of chemokines/chemotactic molecules in response to H 2 O 2 . Shown are means ± SEM. * p < 0.05; * * p < 0.01; * * * p < 0.001. All significances shown are against control unless otherwise indicated. LPS (positive control) was added at 100 ng/ml.
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of marker expression was confirmed with Western blot-ting ( fig. 7 b).
Therefore, H 2 O 2 stimulation of microglia largely elic-ited an M2 polarization on microglial cells with almost all cells being Arginase 1+ and CD206+ by 24 h. There is no expression of M2b markers (IL-1Rα or SOCS3; data not shown).
Discussion
In the present study, exposure of primary microglial cells to continuous, low levels of H 2 O 2 leads to higher LDH release than exposure to higher bolus doses, suggest-ing pleiotropic effects based on mode of H 2 O 2 exposure. ROS levels are significantly elevated by 4 h with bolus
×5000CAT
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15 μM
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Arg1 iNOS4 h 24 h
Arg1 iNOS
Control
a
iNOS
Arginase 1
4 h 24 h
Control LPS LPS×5000 ×500015 μM 15 μMControl
�-Actin
b
Fig. 7. H 2 O 2 exposure elicits a predominantly M2a phenotype in microglia. a Using immunofluorescence with antibodies against M1 (CD32 and iNOS) and M2a (CD206 and Arginase 1) markers, the phenotype of primary microglia in response to H 2 O 2 was char-acterized. For clarity, only iNOS and Arg1 staining are shown here. Although at 4 h microglia are weakly positive for both M1 and M2a markers, by 24 h, they are primarily M2a marker positive. b A Western blot representing M1 and M2a marker protein expres-sion.
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treatment but take longer with continuous exposure, with superoxide anion levels being higher at 4 and 18 h but NO levels detectable only at 24 h. Antioxidant enzyme levels remained largely unchanged, possibly due to insufficient stimulation. At very low but sustained levels, H 2 O 2 poten-tially acts as a preconditioning mechanism eliciting im-mune mediator responses similar to those seen after expo-sure to LPS but largely attenuated. However, unlike LPS, H 2 O 2 promotes the survival/expansion of microglia of an M2 phenotype at later time points, suggesting a largely anti-inflammatory function for surviving microglial cells in response to H 2 O 2 exposure. Together these results sug-gest a potential preconditioning role for low continuous levels of H 2 O 2 in particular, that would enable microglial cells to be ‘primed’ for future insults, with the elimination of the more toxic M1 phenotype.
H 2 O 2 is an important signaling molecule in the brain that, at high doses, acts as an oxidant, causing injury. Giv-en that the antioxidant mechanisms of the immature brain are not as developed as in the adult brain [9] , it is important to characterize cellular responses to such oxi-dative injury. One of the main aims of this study was to employ a method that would expose primary microglial cells to more physiological sustained levels of H 2 O 2 com-pared to that generally employed by studies utilizing bo-lus treatments. The GOX-CAT system enabled this, and, as previously published, at the highest CAT dilution used in this study (×80,000), H 2 O 2 is generated at around 1–5 μ M within a few minutes and is sustained for over 24 h [21] . This system better mimics the sustained rise in H 2 O 2 levels that have been reported after HI in the neonatal brain [17] . Although not specifically tested in our study, it is unlikely that the microglia were oxygen deprived as using a low GOX concentration with minimal medium volume has been established to suffice in averting hypox-ic conditions in this system [21] . The efficacy of the sys-tem is evident from the viability studies where a dose-dependent increase in cell death is seen with increasing CAT dilution (decreasing breakdown of H 2 O 2 ). When compared to bolus treatments, it is obvious that low sus-tained levels of H 2 O 2 elicit a higher LDH release and ap-pear to be more harmful for microglia of the M1 pheno-type. As bolus doses have short half-lives [20, 35] , it is possible that the detrimental effects of H 2 O 2 are attenu-ated due to rapid clearance, especially since microglial cells have significantly high activities of antioxidant en-zymes [32] .
Brain microglial cells make use of the toxic potential of ROS as a defense mechanism. As a result, microglia are known to rapidly produce ROS following activation [27,
28] . In accordance with this, an increase in ROS expres-sion was observed following H 2 O 2 exposure. Since the ROS indicator used in this study is a general oxidative stress indicator, it is possible that most of the ROS de-tected relate to the H 2 O 2 actually applied on the cells es-pecially in the case of bolus doses at 4 h. At 18 and 24 h, however, considerable elevation is seen in superoxide and NO levels suggesting that at least some of the ROS seen are superoxide anions and NO; microglia have been shown to produce both these ROS as products of reac-tions catalyzed by microglial NADPH oxidase and iNOS, respectively [28, 36, 37] . The fact that NO is not seen un-til 24 h is likely related to iNOS protein translation taking up to 24 h although mRNA levels are increased earlier [38] (preliminary analysis in our lab). Interestingly, NO levels were significantly decreased with bolus and ×20,000 CAT treatments when compared to control. NO and su-peroxide together form the highly toxic peroxynitrite [39] and given that significantly high levels of superoxide were seen at 18 h (which reduce by 24 h) at these doses in par-ticular, it is possible that the NO produced at these doses is consumed and depleted. It is possible that at the ×80,000 CAT dose, the extremely high levels of NO in the medium represent a high level of toxicity that results in the consid-erable cell death evident at this dose (and as a result, a decrease in detectable intracellular mitochondrial super-oxide levels).
The high levels of NO at ×80,000 CAT are possibly also responsible for the significantly lower GSH levels at this dose as induction of iNOS has been shown to lower the cellular GSH content in microglial cells [40] . Alternative-ly, it could reflect the extensive cell death seen by 18 h at this dose. Since we do not see large, significant changes in GSSG levels at almost all the doses tested, total GSH mea-sured likely reflects free (reduced) GSH that is able to counteract ROS being produced. Microglial cells have high intracellular concentrations of GSH that contributes strongly to defense against radical- and peroxide-mediat-ed damage [32, 41] . It is possible that at all the doses used in our studies (except the ×80,000 CAT), microglial cells were able to defend themselves from oxidative damage. Indeed, along with GSH, microglial cells have also been shown to express substantial activities of various antioxi-dant enzymes such as superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase, all of which possibly contribute to their self-defense [41] , par-ticularly in vitro where microglia are in isolation. This is supported by the fact that there is some but not extensive cell death at all continuous (except ×80,000 CAT) and bolus doses.
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In addition to production of ROS, microglial cells re-spond to stimulation by releasing cytokines. Engagement of microglial pattern recognition receptors by various stimuli induces signal cascades which, after several hours, produce the chronically activated state of microglia [42] . This is largely due to the expression and release of various proteins, a majority of which are pro- and anti-inflamma-tory cytokines and chemokine/chemotactic molecules. In accordance with this, we observed an increase in various inflammatory mediators particularly at 18 and 24 h. IL-1β, TNF-α and IL-6 have been extensively characterized as immunostimulatory/proinflammatory molecules and have been shown to be elevated early after insult in vari-ous injury models [43–46] , especially in response to LPS stimulation. Here, we did not see a significant rise in their levels in our study using H 2 O 2 stimulation at 4 h. Al-though all other responses were very similar to those seen with LPS, early, significant rise in proinflammatory cyto-kines IL-6, IL-17, TNF-α, and IFN-γ seen with LPS was absent in H 2 O 2 -treated cells. It is possible that at the dos-es tested in this study, H 2 O 2 is not toxic enough to elicit extensive inflammatory cytokine signaling. Alternatively, since microglia have different cytokine responses to dif-ferent stimuli [47, 48] , H 2 O 2 as a stimulus is not a potent inducer of pro- and/or anti-inflammatory cytokines in particular. Of all the doses tested, it is only at the ×5,000 CAT dose that we observed any considerable responses suggesting that at this very low continuous dose, H 2 O 2 is likely acting more as a signaling or preconditioning mol-ecule [22] , while the other continuous as well as bolus doses might be too high for H 2 O 2 to signal as such. The consistent elevation of all factors at 18 and 24 h would then be representative of the effects of prolonged stimula-tion at low doses such as that with ×5,000 CAT.
Significantly high levels of chemokine/chemotactic molecules (G-CSF, MIP-1b, MIP-2) were observed, how-ever, in response to H 2 O 2 exposure. Considering these mediators have been suggested to contribute to ischemic brain injury [49–52] , it is possible that these cytokines contribute to whatever cell death was evident at this dose. However, treatment with or secretion of these chemo-kines promotes the proliferation, migration and activa-tion of more microglial and other cells [53–55] , which in turn would provide support for the containment and res-olution of injury. There is also increasing evidence now to show that chemokines such as MCP-1, MIP-1a, MIP-1b, RANTES, GCSF, GMCSF and MIP-2 are also involved in neuroprotection-regeneration [56–60] . As it appears that the function of microglia (either neuroprotection or neurotoxicity) is determined by the equilibrium among
factors released from activated microglia [4, 61] that we see significantly high levels of almost all of these chemo-kines with low continuous H 2 O 2 , sustained H 2 O 2 might act to ‘prime’ microglia or elicit neuroprotective-regener-ative functions.
The possibility that microglia exposed to low continu-ous H 2 O 2 are potentially neuroprotective is further sup-ported by the finding that exposure to H 2 O 2 promoted an M2a phenotype in microglial cells. Microglial cells have various phenotypes and subtypes with differing function-al properties [3] . M1 microglia are ‘classically activated’ microglia with largely proinflammatory properties while M2a and M2b macrophages/microglia have largely anti-inflammatory and reparative properties. This would sug-gest then that at all doses tested in this study, H 2 O 2 expo-sure serves to promote the survival or expansion of M2a microglia (as seen at 24 h). As we see some expression of M1 (inflammatory) markers at 4 and 18 h, it is likely that M1 microglia are gradually reduced in numbers while M2a microglia survive/expand. Since M2 macrophages are able to promote angiogenesis, downregulate the ex-pression of proinflammatory cytokines [3, 62] as well as elicit other beneficial functions, this would indicate that H 2 O 2 exposure, at least at the continuous and bolus doses tested in this study, would prime these microglia towards a more anti-inflammatory role. It is also encouraging to note that this alternative activation of microglia occurs particularly in the absence of IL-4/IL-13 signaling as IL-4/IL-13 signaling has been shown to be the primary induc-er of an alternative phenotype in microglial cells [47, 63] . Although there is some evidence of microglial IL-4 and IL-13 production in vivo, to our knowledge there is no evidence that IL-4 is produced by isolated microglial cells in vitro [64, 65] .
In summary, although H 2 O 2 is a well-known signaling molecule, we show in our study that nondeleterious levels of H 2 O 2 , especially when sustained, can act as a potential preconditioning mechanism, promoting microglia of the anti-inflammatory/wound-healing phenotype. Techni-cally, this would mean that in the immature brain where there is a sustained increase in H 2 O 2 levels following HI injury, microglia at least might be able to survive and pro-vide protection/regeneration. However, since injury is prevalent and occurs rapidly, it is likely that the levels are much higher following injury and as such, as seen with our continuous higher doses, renders microglia suscep-tible to injury. It is also important to recognize that mi-croglial cell cultures in isolation can behave very differ-ently to co-cultures or microglia in vivo. Hence, although findings reported here provide important information on
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microglial responses in isolation without the confound-ing effects by other cell types, they should be interpreted with caution and related to co-culture or in vivo studies wherever possible.
Acknowledgement
This work was supported by funding from Fondation LeDucq 10 CVD-01.
References
1 Hagberg H, Gilland E, Bona E, Hanson LA, Hahin-Zoric M, Blennow M, et al: Enhanced expression of interleukin (IL)-1 and IL-6 mes-senger RNA and bioactive protein after hy-poxia-ischemia in neonatal rats. Pediatr Res 1996; 40: 603–609.
3 David S, Kroner A: Repertoire of microglial and macrophage responses after spinal cord injury. Nat Rev Neurosci 2011; 12: 388–399.
4 Li L, Lu J, Tay SSW, Moochhala SM, He BP: The function of microglia, either neuropro-tection or neurotoxicity, is determined by the equilibrium among factors released from ac-tivated microglia in vitro. Brain Res 2007; 1159: 8–17.
5 Alvarez-Díaz A, Hilario E, de Cerio FG, Vall-s-i-Soler A, Alvarez-Díaz FJ: Hypoxic-isch-emic injury in the immature brain – key vascu lar and cellular players. Neonatology 2007; 92: 227–235.
6 Ferriero DM: Oxidant mechanisms in neona-tal hypoxia-ischemia. Dev Neurosci 2001; 23: 198–202.
7 Halliwell B: Reactive oxygen species and the central nervous system. J Neurochem 1992; 59: 1609–1623.
9 Mischel RE, Kim YS, Sheldon RA, Ferriero DM: Hydrogen peroxide is selectively toxic to immature murine neurons in vitro. Neurosci Lett 1997; 231: 17–20.
10 Fan L-W, Pang Y, Lin S, Rhodes PG, Cai Z: Minocycline attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain. Neuroscience 2005; 133: 159–168.
11 Filipovic R, Zecevic N: Neuroprotective role of minocycline in co-cultures of human fetal neurons and microglia. Exp Neurol 2008; 211: 41–51.
12 Lechpammer M, Manning SM, Samonte F, Nelligan J, Sabo E, Talos DM, et al: Minocy-cline treatment following hypoxic/ischaemic injury attenuates white matter injury in a ro-dent model of periventricular leucomalacia. Neuropathol Appl Neurobiol 2008; 34: 379–393.
13 Arvin KL, Han BH, Du Y, Lin S, Paul SM, Holtzman DM: Minocycline markedly pro-tects the neonatal brain against hypoxic-isch-emic injury. Ann Neurol 2002; 52: 54–61.
14 Tsuji M, Wilson MA, Lange MS, Johnston MV: Minocycline worsens hypoxic-ischemic brain injury in a neonatal mouse model. Exp Neurol 2004; 189: 58–65.
15 Faustino JV, Wang X, Johnson CE, Klibanov A, Derugin N, Wendland MF, et al: Microg-lial cells contribute to endogenous brain de-fenses after acute neonatal focal stroke. J Neu-rosci 2011; 31: 12992–13001.
16 Lalancette-Hébert M, Gowing G, Simard A, Weng YC, Kriz J: Selective ablation of prolif-erating microglial cells exacerbates ischemic injury in the brain. J Neurosci 2007; 27: 2596–2605.
17 Lafemina MJ, Sheldon RANN, Ferriero DM, L DNMJ, Pediatrics DMF, San C: Acute hy-poxia-ischemia results in hydrogen peroxide accumulation in neonatal but not adult mouse brain. Pediatr Res 2006; 59: 680–683.
18 Mueller S, Arnhold J: Fast and sensitive che-miluminescence determination of H 2 O 2 con-centration in stimulated human neutrophils. J Biolumin Chemilumin 1995; 10: 229–237.
19 Müller S: Sensitive and nonenzymatic mea-surement of hydrogen peroxide in biological systems; in Pryor WA (ed): Bio-Assays for Oxidative Stress Status (BOSS). New York, El-sevier, 2001, pp 170–175.
20 Gülden M, Jess A, Kammann J, Maser E, Sei-bert H: Cytotoxic potency of H 2 O 2 in cell cul-tures: impact of cell concentration and expo-sure time. Free Radic Biol Med 2010; 49: 1298–1305.
21 Mueller S, Millonig G, Waite GN: The GOX/CAT system: a novel enzymatic method to in-dependently control hydrogen peroxide and hypoxia in cell culture. Adv Med sci 2009; 54: 121–135.
22 Chang S, Jiang X, Zhao C, Lee C, Ferriero DM: Exogenous low dose hydrogen peroxide in-creases hypoxia-inducible factor-1alpha pro-tein expression and induces preconditioning protection against ischemia in primary corti-cal neurons. Neurosc Lett 2008; 441: 134–138.
23 Millonig G, Hegedüsch S, Becker L, Seitz H, Schuppan D, Mueller S: Hypoxia-inducible factor 1α under rapid enzymatic hypoxia: cells sense decrements of oxygen but not hypoxia per se. Free Rad Biol Med 2009; 46: 182–191.
24 Dean JM, Wang X, Kaindl AM, Gressens P, Fleiss B, Hagberg H, et al: Microglial MyD88 signaling regulates acute neuronal toxicity of LPS-stimulated microglia in vitro. Brain Be-hav Immun 2010; 24: 776–783.
25 Rahman I, Kode A, Biswas SK: Assay for quantitative determination of glutathione and glutathione disulfide levels using enzy-matic recycling method. Nat Protoc 2006; 1: 3159–3165.
26 Mueller S, Weber A, Fritz R, Mutze S, Rost D, Walczak H, Bolkl A SW: Sensitive and real-time determination of H 2 O 2 release from intact per-oxisomes. Biochem J 2001; 363: 483–491.
27 Streit WJ: Microglia as neuroprotective, im-munocompetent cells of the CNS. Exp Neurol 2002; 139: 133–139.
28 Minghetti L, Levi G: Microglia as effector cells in brain damage and repair: focus on pros-tanoids and nitric oxide. Prog Neurobiol 1998; 54: 99–125.
29 Liu Y, Schubert DR: The specificity of neuro-protection by antioxidants. J Biomed Sci 2009; 16: 98.
30 LeBel CP, Ischiropoulos H, Bondy SC: Evalu-ation of the probe 2′,7′-dichlorofluorescin as an indicator of reactive oxygen species forma-tion and oxidative stress. Chem Res Toxicol 1992; 5: 227–231.
31 Robinson KM, Janes MS, Beckman JS: The se-lective detection of mitochondrial superoxide by live cell imaging. Nat Protoc 2008; 3: 941–947.
32 Hirrlinger J, Gutterer JM, Kussmaul L, Ham-precht B, Dringen R: Microglial cells in cul-ture express a prominent glutathione system for the defense against reactive oxygen spe-cies. Dev Neurosci 2000; 22: 384–392.
33 Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG: Identification of two distinct macrophage subsets with di-vergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 2009; 29: 13435–13444.
34 Auffray C, Sieweke MH, Geissmann F: Blood monocytes: development, heterogeneity, and relationship with dendritic cells. Ann Rev Im-munol 2009; 27: 669–692.
Dev Neurosci 2013;35:241–254DOI: 10.1159/000346159
254
37 Sankarapandi S, Zweier JL, Mukherjee G, Quinn MT, Huso DL: Measurement and characterization of superoxide generation in microglial cells: evidence for an NADPH oxi-dase-dependent pathway. Arch Biochem Bio-phys 1998; 353: 312–321.
38 Ding M, St Pierre BA, Parkinson JF, Medber-ry P, Wong JL, Rogers NE, et al: Inducible ni-tric-oxide synthase and nitric oxide produc-tion in human fetal astrocytes and microglia. A kinetic analysis. J Biol Chem 1997; 272: 11327–11335.
39 Bal-Price A, Matthias A, Brown GC: Stimula-tion of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production. J Neurochem 2002; 80: 73–80.
40 Chatterjee S, Noack H, Possel H, Wolf G: In-duction of nitric oxide synthesis lowers intra-cellular glutathione in microglia of primary glial cultures. Glia 2000; 29: 98–101.
41 Dringen R: Oxidative and antioxidative po-tential of brain microglial cells. Antioxid Re-dox Signal 2005; 7: 1223–1233.
42 Brown GC, Neher JJ: Inflammatory neurode-generation and mechanisms of microglial killing of neurons. J Neurosci Res 2010; 41: 242–247.
43 Simi A, Tsakiri N, Wang P, Rothwell NJ: In-terleukin-1 and inflammatory neurodegen-eration. Biochem Soc Trans 2007; 35: 1122–1126.
44 McCoy MK, Tansey MG: TNF signaling inhi-bition in the CNS: implications for normal brain function and neurodegenerative dis-ease. J Neuroinflammation 2008; 5: 45.
45 Raivich G, Jones LL, Werner A, Blüthmann H, Doetschmann T, Kreutzberg GW: Molecu-lar signals for glial activation: pro- and anti-inflammatory cytokines in the injured brain. Acta Neurochir Suppl 1999; 73: 21–30.
46 Rothwell N, Allan S, Toulmond S: The role of interleukin 1 in acute neurodegeneration and stroke: pathophysiological and therapeutic implications. J Clin Invest 1997; 100: 2648–2652.
47 Suzumura A: Immune response in the brain: glial response and cytokine production; in Phelps C, Korneva E (eds): Cytokines and the Brain. Amsterdam, Elsevier, 2008, pp 289–303.
48 Olah M, Biber K, Vinet J, Boddeke HWGM: Microglia phenotype diversity. CNS Neurol Disord Drug Targets 2011; 10: 108–118.
49 Terao S, Yilmaz G, Stokes KY, Russell J, Ishikawa M, Kawase T, et al: Blood cell-de-rived RANTES mediates cerebral microvas-cular dysfunction, inflammation, and tissue injury after focal ischemia-reperfusion. Stroke 2008; 39: 2560–2570.
50 Dimitrijevic OB, Stamatovic SM, Keep RF, Andjelkovic AV: Effects of the chemokine CCL2 on blood-brain barrier permeability during ischemia-reperfusion injury. J Cereb Blood Flow Metab 2006; 26: 797–810.
51 Dimitrijevic OB, Stamatovic SM, Keep RF, Andjelkovic AV: Absence of the chemokine receptor CCR2 protects against cerebral isch-emia/reperfusion injury in mice. Stroke 2007; 38: 1345–1353.
53 Belmadani A, Tran PB, Ren D, Miller RJ: Che-mokines regulate the migration of neural pro-genitors to sites of neuroinflammation. J Neu-rosci 2006; 26: 3182–3191.
55 Bartolini A, Vigliani M-C, Magrassi L, Ver-celli A, Rossi F: G-CSF administration to adult mice stimulates the proliferation of microglia but does not modify the outcome of ischemic injury. Neurobiol Dis 2011; 41: 640–649.
56 Bruno V, Copani A, Besong G, Scoto G, Nico-letti F: Neuroprotective activity of chemo-kines against N-methyl- D -aspartate or beta-amyloid-induced toxicity in culture. Eur J Pharmacol 2000; 399: 117–121.
57 Eugenin EA, D’Aversa TG, Lopez L, Calderon TM, Berman JW: MCP-1 (CCL2) protects hu-man neurons and astrocytes from NMDA or HIV-tat-induced apoptosis. J Neurochem 2003; 85: 1299–1311.
58 Chiu K, Yeung S-C, So K-F, Chang RC-C: Modulation of morphological changes of mi-croglia and neuroprotection by monocyte chemoattractant protein-1 in experimental glaucoma. Cell Mol Immunol 2010; 7: 61–68.
59 Yamasaki R, Tanaka M, Fukunaga M, Tatei-shi T, Kikuchi H, Motomura K, et al: Restora-tion of microglial function by granulocyte-colony stimulating factor in ALS model mice. J Neuroimmunol 2010; 229: 51–62.
60 Esen N, Kielian T: Effects of low dose GM-CSF on microglial inflammatory profiles to diverse pathogen-associated molecular pat-terns (PAMPs). J Neuroinflammation 2007; 4: 10.
61 Suzumura A, Takeuchi H, Zhang G, Kuno R, Mizuno T: Roles of glia-derived cytokines on neuronal degeneration and regeneration. Ann NY Acad Sci 2006; 1088: 219–229.
62 Sica A, Larghi P, Mancino A, Rubino L, Porta C, Totaro MG, et al: Macrophage polarization in tumour progression. Seminars Cancer Biol 2008; 18: 349–355.
63 Martinez FO, Helming L, Gordon S: Alterna-tive activation of macrophages: an immuno-logic functional perspective. Ann Rev Immu-nol 2009; 27: 451–483.
64 Suzumura A, Sawada M, Itoh Y, Marunouchi T: Interleukin-4 induces proliferation and ac-tivation of microglia but suppresses their in-duction of class II major histocompatibility complex antigen expression. J Neuroimmu-nol 1994; 53: 209–218.
65 Microglia expressing interleukin-13 undergo cell death and contribute to neuronal survival in vivo. 2012. http://www.abd.or.kr/htm/jin/26.pdf.
