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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2015 May 13;35(10):1623–1631. doi: 10.1038/jcbfm.2015.97

CX3CL1/CX3CR1-mediated microglia activation plays a detrimental role in ischemic mice brain via p38MAPK/PKC pathway

Yong Liu 1,5, Xiao-Mei Wu 2,3,5, Qian-Qian Luo 2,4, Suna Huang 4, Qing-Wu Qian Yang 1, Fa-Xiang Wang 1, Ya Ke 3,*, Zhong-Ming Qian 2,4,*
PMCID: PMC4640309  PMID: 25966946

Abstract

The exact roles of activated microglia and fractalkine (CX3CL1)/fractalkine receptor (CX3CR1) signaling are not fully understood in brain ischemic injury and the findings reported are controversial. Here, we investigated the effects of CX3CR1 siRNA on the expression of CX3CR1, p38 mitogen-activated protein kinase (p38MAPK), Protein Kinase C (PKC) and inflammatory cytokines, microglia activation, white matter lesions, and cognitive function in mice treated with bilateral common carotid artery stenosis (BCAS) in vivo as well as effects of exogenous CX3CL1, CX3CR1 siRNA, and SB2035080 on expression of inflammatory cytokines in BV2 microglia treated with oxygen–glucose deprivation (OGD) in vitro. We showed that CX3CR1 siRNA significantly inhibited the increased expression of CX3CR1, p38MAPK, PKC as well as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, and also attenuated microglia activation, white matter lesions, and cognitive deficits induced by BCAS in mice brain. We also showed that exogenous CX3CL1 could induce a further enhancement in TNF-α and IL-1β expression, which could be suppressed by CX3CR1 siRNA or by the p38MAPK inhibitor in OGD-treated BV2 microglial cells in vitro. Our findings indicated that CX3CL1/CX3CR1-mediated microglial activation plays a detrimental role in ischemic brain via p38MAPK/PKC signaling and also suggested that CX3CL1/CX3CR1 axis might be a putative therapeutic target to disrupt the cascade of deleterious events that lead to brain ischemic injury.

Keywords: bilateral common carotid artery stenosis (BCAS), brain ischemic injury, CX3CL1 (fractalkine)/CX3CR1, microglia activation, oxygen–glucose deprivation (OGD), p38MAPK/PKC

Introduction

Microglia is key modulators of the immune response in the brain.1 Under normal physiologic conditions, these cells constantly survey their microenvironment for noxious agents and injurious processes,2 respond to extracellular signals and are responsible for clearing debris and toxic substances by phagocytosis, thereby maintaining normal cellular homeostasis in the central nervous system.3 Under the pathologic circumstance, these cells could be activated at very early stage.4 In animal models of cerebral ischemia, the processes of microglial activation have been studied extensively, however, the exact function of these activated cells is not fully understood5 and the findings reported are controversial.6, 7, 8 The answers to the question of whether activated microglial responses are destructive or beneficial after ischemic injury remains equivocal.4, 8

Microglial activation is usually regulated by the chemokine fractalkine (CX3CL1) CX3CL1 and its receptor, CX3CR1.4 CX3CL1 is a relatively new member of the chemokine (chemotactic cytokine) family and the sole member of the CX3C chemokine class,9, 10 which exists in both membrane bound and soluble forms.9 In contrast to many other chemokines, CX3CL1 binds only one receptor CX3CR1.11 In the brain, CX3CL1 is a unique chemokine, being constitutively expressed by neurons where it is tethered to the extracellular membrane by a mucin stalk.9, 12 The CX3CL1 receptor CX3CR1 is a G-protein-coupled receptor and exclusively expressed by microglia.13 The CX3CL1/CX3CR1 signaling pathway has been shown to play an important role in the maintenance of neural–immune communication and the bidirectional interaction between neurons and microglia in health and disease.14, 15

A number of studies have been conducted to investigate the role of CX3CL1/CX3CR1 signaling in brain ischemic injury, however, the relevant findings are also controversial. The destructive or beneficial roles of CX3CL1/CX3CR1 in brain ischemic injury have also been reported. Mice deficient in CX3CL1 were found to be less susceptible to cerebral ischemia–reperfusion injury when compared with wild-type littermates.16 And lack or deficiency of CX3CR1 was shown to reduce significantly ischemic damage and inflammation in mice with focal cerebral ischemia17 and to suppress activation and neurotoxicity of microglia/macrophage in experimental ischemic stroke.18 In addition, CX3CL1- and CX3CR1-knockout mice revealed to have less severe brain injury on permanent19 and transient20 middle cerebral artery occlusion. These studies suggested that CX3CL1–CX3CR1 expression is detrimental to recovery after ischemic injury.15, 16 However, it has also been reported that CX3CR1 deficiency worsens the behavioral impairment induced by transient global cerebral ischemic injury and silencing CX3CR1 expression exacerbates the learning and memory deficits.4 Moreover, administration of exogenous CX3CL1 was found to reduce ischemia-induced cerebral infarct size and neurologic deficits in in vivo murine models of permanent middle cerebral artery occlusion.19 These conflicting data to date do not provide a coherent conclusion on the role of CX3CL1/CX3CR1 in brain injury and disease.4, 20

To further explore the roles of this signaling pathway and microglial activation in brain ischemic injury, we investigated the effects of CX3CR1 siRNA (silencing CX3CR1 expression) on expression of CX3CR1, p38 mitogen-activated protein kinase (p38MAPK), protein kinase C (PKC), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, and also microglia activation, white matter lesions, and cognitive function in the brain in mice model of bilateral common carotid artery stenosis (BCAS) in vivo. We also examined the effects of the addition of exogenous CX3CL1 with CX3CR1 siRNA and SB2035080 (SB, a p38MAPK inhibitor) on the expression of TNF-α and IL-1β in OGD (oxygen–glucose deprivation) treated BV2 microglial cells in vitro. The p38MAPK was chosen to be examined because this signaling molecule has been well documented to be associated with the expression of inflammatory and cytotoxic mediators.21 Our findings indicated that CX3CL1, acting on the CX3CR1 of microglia, activates p38MAPK/PKC and promotes the generation of TNF-α and IL-1β inducing a detrimental effect in the brain of ischemic mice under our experimental conditions.

Materials and Methods

Materials

Unless otherwise stated, all chemicals were obtained from Sigma Chemical Company, St. Louis, MO, USA. Rabbit polyclonal TNF-α was purchased from Affiniti Research, Devon, UK, and rabbit monoclonal IL-1β and rabbit polyclonal IL-6 from Lab Vision Corporation, Fremont, CA, USA. Rabbit polyclonal anti-CX3CR1 was obtained from Merck Millipore, Billerica, MA, USA, antibodies against p38MAPK and PKC were purchased from CST, Cell Signaling Technology, Danvers, MA, USA and TNF-α and IL-1β ELISA (enzyme-linked immunosorbent assay) kits from R&D Systems China, Shanghai, PRC. Sheep antirat biotinylated IgG, antirabbit secondary antibody–conjugated horseradish peroxidase (HRP), and HRP–streptavidin were obtained from Zhongshan Biotech, Beijing, China; rat monoclonal anti-CD11b antibody from AbD Serotec, Kidlington, UK; and Bradford assay kit from Bio-Rad, Hercules, CA, USA. CX3CR1 siRNA (against CX3CR1 retrovirus) and negative siRNA (negative control) were obtained from GeneChem, Shanghai, PRC.

Animals, BV2 Microglia, and Primary Cultured Neurons

Male C57BL/6J mice (8 to 10 weeks) weighing 20 to 24 g were supplied by the Animal Center of the Third Military Medical University (Chongqing, China) and housed in individual cages under a standard 12-hour light–dark cycle with water and food supplied ad libitum. All animal handling and surgical procedures were approved by the Animal Research Ethics Committee of the Third Military Medical University in accordance with the guidelines of the Chongqing City Health Department on Animal Care. The experiments were performed in accordance with the Animal Research: Reporting In Vivo Experiments guidelines.

BV2 microglial cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco) and 100 μg/mL penicillin–streptomycin (Gibco) at 37°C in humidified atmosphere containing 5% CO2.22 The primary cortical neuronal culture was prepared from embryonic day 14 to 15 C57BL/6J mice using a method as described previously.23

Experimental design

In vivo study

Two-vessel occlusion caused tissue injury in C57BL/6 mice, which is highly variable because of the anatomic variations of the posterior communicating artery.24, 25, 26 We therefore premeasured cerebral blood flow before and 2 hours after BCAS with laser-Doppler flowmetry as described previously27 to exclude animals that are less susceptible (cerebral blood flow 2 hours after BCAS/cerebral blood flow before ⩾65%) to ischemic insult. To determine the role of CX3CL1/CX3CR1-mediated activation of microglia and the relevant mechanisms under the conditions of ischemia, a total of 27 susceptible C57BL/6J mice were randomly assigned into three groups. The mice in BCAS or BCAS+siRNA group were pretreated with 3 μL of saline (BCAS group, n=9) or CX3CR1 siRNA (BCAS+siRNA group, n=11) by intracerebroventricular injection, and five days later, underwent with BCAS. The animals in the sham-operated group (n=7) underwent the same surgical procedure as the above two groups without BCAS. At day 14 after BCAS, the expression of CX3CR1, p38MAPK, and PKC, and the contents of TNF-α, IL-1β, and IL-6 were determined by Western blot assay, and also microglia activation and white matter lesions were evaluated by histochemical method, cognitive function assessed by water maze test, and the expression of CX3CR1 was also assessed by immunostain analysis.

In vitro study

To further explore the relevant mechanisms involved in the role of fractalkine/CX3CR1-mediated activation of microglia under ischemia, the BV2 microglial cells were treated with Oxygen–glucose deprivation (OGD) in the presence of CX3CL1 (0, 12.5, 25, and 50 ng/mL), or infected with 10 Multiplicity of Infection (MOI) CX3CR1 siRNA or negative siRNA for 72 hours or pretreated with 20 μmol/L of SB2035080 only for 1 hour, before subjected to OGD for 4 hours in the presence of 0 or 50 ng/mL of CX3CL1. To investigate whether neurons prepared from C57BL/6J mice could secrete and express CX3CL1 and whether OGD affect the release and expression of CX3CL1 in neurons, neurons were treated with OGD for 4 hours and then reperfusion (R) for 24 hours. The contents of CX3CL1 in neuronal medium was detected by ELISA and the expression of CX3CL1 in neurons were observed by immunostaining. OGD was performed by exposing the cells to serum-free DMEM without glucose in a hypoxic incubator (INVIVO2, RUSKINN, Sanford, Maine, USA) with 1% O2, 94% N2, and 5% CO2 at 37°C for 4 hours as previously described.28 After the treatments, the measurements of TNF-α, IL-1β, and CX3CL1 were conducted using ELISA.

Intracerebroventricular Injection

Injections (intracerebroventricular) were accomplished via an indwelling guide cannula stereotaxically implanted into the right lateral cerebral ventricle (bregma −0.58 mm, lateral 1.20 mm, and depth 2.25 mm).28

Bilateral Common Carotid Artery Stenosis

Male C57BL/6J mice were subjected to BCAS, which was performed by applying the microcoils (Sawane Spring, Osaka, Japan) with an inner diameter of 0.18 mm to common carotid arteries (CCAs).26 Briefly, both CCAs were exposed and freed from their sheaths, through a midline cervical incision, after the mice were intraperitoneally anesthetized with sodium pentobarbital (50 mg/kg). Two 4 to 0 silk sutures were placed around the distal and proximal parts of the right CCA. Then, the artery was gently lifted by these sutures and placed between the loops of the microcoil just below the carotid bifurcation. The microcoil was twined by rotating it around the CCA. After 30 minutes, another microcoil of the same size was twined around the left CCA. The rectal temperature was maintained between 36.5°C and 37.5°C. Sham-operated mice underwent the same surgical procedure without using coils.

Western Blot Assay

The expression of CX3CR1, p38MAPK, PKC, TNF-α, IL-1β, and IL-6 in brain tissue of the mice were determined as described previously.29 Total protein content was determined using the Bradford assay kit (Bio-Rad). The blots were probed with primary antibodies: rabbit polyclonal anti-CX3CR1 (1:1,000), p38MAPK (1:1,000), PKC (1:1,000), rabbit polyclonal TNF-α (1:500), rabbit monoclonal IL-1β (1:1,000), and rabbit polyclonal IL-6 (1:1,000), and then antirabbit secondary antibody–conjugated horseradish peroxidase (1:2,500). The intensity of the specific bands was detected and analyzed by Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln NE, USA). To ensure even loading of the samples, the same membrane was probed with rabbit antirat β-actin polyclonal antibody at a 1:2,000 dilution.

Enzyme-Linked Immunosorbent Assay

TNF-α and IL-1β concentrations in the culture medium of BV2 microglia and CX3CL1 in the culture medium of neurons were determined using commercially available ELISA kits according to the manufacturer's instruction (R&D Systems, China). The optical density at 450 nm was read by using an ELX-800 microplate assay reader (Elx800, Bio-tek, Winooski, VT, USA). The average absorbance values for each set of standards and samples were calculated from the standard curve.30

Immunohistochemistry

Coronal sections (30 μm) were treated with 3% H2O2 in 0.01 mol/L phosphate-buffered saline and preincubated in 5% normal goat serum. Sections were then incubated with the primary antibody, rat monoclonal anti-CD11b antibody (1:100) at 4°C overnight, then with sheep antirat biotinylated IgG (1:200) for 1 hour at room temperature and finally incubated in HRP–streptavidin (1:200) for 1 hour at room temperature. The color reaction was conventionally developed with 3,3′-diaminobenzidine and H2O2.

White Matter Lesion Evaluation

At day 14 after BCAS, the brains were removed and postfixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB) (pH 7.4) for 12 hour, and then stored in 30% sucrose in 0.1 mol/L PB (pH 7.4). Serial coronal sections were cut on a cryostat, spanning from the anterior aspect of the corpus callosum (bregma 0.26 mm) to the anterior aspect of the hippocampus (bregma −0.58 mm). Every fifth section (180 μm) was cut at 10 μm and processed for Klüver-Barrera staining. The severity of the lesions in the corpus callosum was graded as normal (grade 0), disarrangement of the nerve fibers (grade 1), formation of marked vacuoles (grade 2), or disappearance of myelinated fibers (grade 3).26

Water Maze Test

The cognitive function was assessed using a Morris water maze paradigm at day 14 after BCAS.31 The testing was conducted in five consecutive days. Each mouse was subjected to eight trials per day with a 5-minute break between trials. A white and circular pool (1.5-m diameter and 45-cm deep) was filled with water to a 25-cm depth. Water temperature was maintained at ~22°C. A clear Plexiglas platform (11 × 12 cm) was positioned 1 cm below the surface of water. Mice were placed in the tank facing the wall, randomizing to one of four starting locations (north, south, east, or west), and given 90 seconds to find the platform, mount the platform, and remain on it for 5 seconds. The mice were then placed under a heat lamp to dry before their next trial. The time until the mouse mounted the platform (escape latency) was measured and recorded. The data of Morris water maze test were analyzed by the repeated measures analysis of variance.

Statistical Analysis

Statistical analyses were performed using SPSS software for Windows (version 10.0; SPSS, Chicago, IL, USA). Data were presented as mean±s.e.m. The difference between or among the means was determined by Kruskal–Wallis test followed by Mann–Whitney test for multiple comparisons or one-way or two-way analysis of variance in appropriate experiments followed by Newman–Keuls post hoc test. A probability value of P<0.05 was considered to be statistically significant.

Results

CX3CR1 siRNA Significantly Inhibits the Increased Expression of CX3CR1 as well as p38MAPK and PKC Induced by Bilateral Common Carotid Artery Stenosis in Mice Brain In Vivo

To determine the role of CX3CL1/CX3CR1-mediated activation of microglia and the relevant mechanisms under ischemia, we initially investigated the effects of BCAS on the expression of CX3CR1 and also p38MAPK and PKC in mice brain. Western blot analysis showed that the contents of CX3CR1 as well as p38MAPK and PKC in the brain of mice treated with BCAS all were significantly higher than those of mice in sham-operated group (Figures 1A and 1B). Immunostain analysis also showed that BCAS could significantly increase CX3CR1 expression (Figure 1C). These evidenced that ischemia induced by BCAS could induce a significant increase in the expression of the receptor and molecules involved in the signal transduction pathway. We then observed the effects of CX3CR1 siRNA on the expression of CX3CR1, p38MAPK, and PKC in the brain of the ischemic mice to find out whether the increased expression of p38MAPK and PKC is associated with the expression of CX3CR1. We showed that the levels of CX3CR1, p38MAPK, and PKC in the mice treated with CX3CR1 siRNA and then BCAS were markedly lower than the corresponding values in the mice treated with BCAS only (Figures 1A and 1C). The finding indicated that the reduced expression of CX3CR1 induced by CX3CR1 siRNA could lead to a remarkable reduction in the expression of p38MAPK and PKC in the brain of BCAS mice and evidenced that the significant increase in the expression of p38MAPK and PKC induced by BCAS is CX3CR1 dependent in mice brain.

Figure 1.

Figure 1

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CX3CR1 siRNA significantly inhibits the increased expression of CX3CR1, p38MAPK, and PKC, and microglia activation induced by bilateral common carotid artery stenosis (BCAS) in mice brain in vivo. The mice in BCAS (n=9) or BCAS+siRNA (n=11) group were pretreated with saline or CX3CR1 siRNA, and then underwent with BCAS. The animals in the sham-operated group (n=7) underwent the same surgical procedure as the above two groups without BCAS. At day 14 after BCAS, the expression of CX3CR1, p38MAPK, and PKC (A and B) was determined by Western blot assay. The expression of CX3CR1 was also assessed by immunostain analysis (C) and microglia activation was evaluated by histochemical method using rat monoclonal anti-CD11b antibody (D), and the data were quantified respectively (E, CX3CR1; F, anti-CD11b). Scale bar, 50 μm in C and D. Data were represented as mean±s.e.m. *P<0.05 versus sham; #P<0.05 versus BCAS. IOD, integrated optical density; OD, optical density.

CX3CR1 siRNA Significantly Inhibits Microglia Activation Induced by Bilateral Common Carotid Artery Stenosis in Mice Brain In Vivo

Second, we investigated whether the ischemia induced by BCAS could activate microglia by immunohistochemical analysis using CD11b, which was used to identify ischemia-induced activation of microglia.32 Immunohistochemical data (Figure 1D) revealed that microglia have swollen and larger cell bodies with thicker and shorter processes that form thick bundles around an enlarged cell body or a large cell body with almost absent processes in the brain of mice treated with BCAS, indicating that BCAS could lead to activation of microglia. We then evaluated the effects of CX3CR1 siRNA (inhibiting CX3CR1 expression) on microglia activation induced by BCAS to understand whether the activation is related to the expression of CX3CR1. We showed that the morphologic changes found in microglia in the mice treated with BCAS disappeared mostly in the brain of the mice treated with CX3CR1 siRNA and then BCAS. The findings evidenced that treatment with CX3CR1 siRNA could significantly attenuate the BCAS-induced microglia activation and also implied that CX3CR1 played a key and essential role in BCAS-induced microglia activation in mice brain.

CX3CR1 siRNA Significantly Inhibits the Increased Expression of Tumor Necrosis Factor-α, Interleukin-1β, and Interleukin-6 in Mice Brain In Vivo

Third, we examined the effects of ischemia induced by BCAS on the expression of microglia cytokines, including TNF-α, IL-1β, and IL-6, in mice brain. Western blot analysis showed that the contents of TNF-α as well as IL-1β and IL-6 proteins in the brain of mice treated with BCAS were significantly higher than those of mice in the sham-operated group (Figures 2A and 2B). This showed that ischemia could induce a remarkable increase in the expression of these microglia cytokines. We then explored the effects of CX3CR1 siRNA on the expression of TNF-α, IL-1β, and IL-6 in the brain of the ischemic mice to find out whether the increased expression of TNF-α, IL-1β, and IL-6 is associated with the expression of CX3CR1. We showed that the levels of TNF-α, IL-1β, and IL-6 in the mice treated with CX3CR1 siRNA and then BCAS were significantly lower than those in the mice treated with BCAS only. The finding suggested that the reduced expression of CX3CR1 induced by CX3CR1 siRNA could lead to a remarkable reduction in the expression of TNF-α, IL-1β, and IL-6 in the brain of BCAS mice and evidenced that the significant increase in the expression of these microglia cytokines induced by BCAS is also CX3CR1 dependent in mice brain.

Figure 2.

Figure 2

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CX3CR1 siRNA significantly reduces the increased expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 in mice brain in vivo. The mice in bilateral common carotid artery stenosis (BCAS, n=9) or BCAS+siRNA (n=11) group were pretreated with saline or CX3CR1 siRNA by intracerebroventricular injection, and then treated with BCAS. The animals in the sham-operated group (n=7) underwent the same surgical procedure as the above two groups without BCAS. At day 14 after BCAS, the expression of TNF-α, IL-1β, and IL-6 was determined by Western blot assay. (A) A representative experiment of Western blot. (B) Quantification of expression of proteins. Data were represented as mean±s.e.m. *P<0.05 versus sham; #P<0.05 versus BCAS. OD, optical density.

CX3CR1 siRNA Significantly Attenuates White Matter Lesions and Cognitive Deficits Induced by Bilateral Common Carotid Artery Stenosis in Mice

Finally, we examined the effects of BCAS and CX3CR1 siRNA on white matter lesion and cognitive function by histochemical method and water maze test. Histochemical analysis (Figures 3A and 3C) showed that the white matter rarefaction grading scores in mice of the BCAS group were significantly higher than those in mice of the sham-operated group. However, the scores in mice in the BCAS group were significantly lower than those in mice in the BCAS+siRNA group. The findings implied that BCAS could lead to white matter lesion, while CX3CR1 siRNA could dramatically suppress the lesion in mice. The results obtained from water maze test (Figure 3B) showed that the mean escape latency in mice of the BCAS group were significantly longer than those in mice of the sham-operated group. However, the mean escape latency in mice in the BCAS group were significantly shorter than those in mice in the BCAS+siRNA group. The data indicated that BCAS could lead to cognitive deficits, while CX3CR1 siRNA could significantly attenuate the deficits in mice. The findings also showed that CX3CR1-associated microglia activation plays a detrimental role in ischemic mice under our experimental conditions.

Figure 3.

Figure 3

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CX3CR1 siRNA significantly attenuates white matter lesions and cognitive deficits induced by bilateral common carotid artery stenosis (BCAS) in mice. The mice in BCAS (n=9) or BCAS+siRNA (n=11) group were pretreated with saline or CX3CR1 siRNA by intracerebroventricular injection, and then underwent BCAS. The animals in the sham-operated group (n=7) underwent the same surgical procedure as the above two groups without BCAS. At day 14 after BCAS, grading scores=white matter rarefaction grading scores (A) were evaluated by histochemical method, and cognitive function, Mean Escape latency (seconds)=mean escape latency (seconds; B) assessed by water maze test. (C) A representative histologic analysis of white matter lesion. Data were represented as mean±s.e.m. *P<0.05 versus sham; #P<0.05 versus BCAS.

CX3CL1 Induced a Further Enhancement in Tumor Necrosis Factor-α and Interleukin-1β Expression, Which Could be Significantly Attenuated by CX3CR1 siRNA or the p38MAPK Inhibitor in Oxygen–Glucose Deprivation–Treated BV2 Microglial Cells In Vitro

To further investigate the relevant mechanisms involved in the detrimental role of CX3CR1-associated activation of microglia under ischemia, we then investigated the effects of exogenous CX3CL1 with CX3CR1 siRNA and SB2035080 on the expression of microglia cytokines TNF-α and IL-1β in OGD-treated BV2 microglial cells in vitro. CX3CL1 was used in this study because its role in the CX3CR1-associated activation of microglia was well demonstrated.11 The findings (Figures 4A and 4B) showed that the levels of TNF-α and IL-1β in the culture medium were significantly higher in OGD-treated BV2 microglia than in control cells, indicating that OGD could induce a significant increase in TNF-α and IL-1β release from BV2 microglia. Also, the addition of different concentrations (0, 12.5, 25, or 50 ng/mL) of CX3CL1 led to a progressive increase in the contents of TNF-α and IL-1β in the BV2 microglial culture medium (Figures 4C and 4D), implying that CX3CL1 has a role to increase TNF-α and IL-1β expression in BV2 microglia treated without OGD. In addition, the treatment with OGD plus 50 ng/mL of CX3CL1 induced a more significant increase in the expression of TNF-α and IL-1β in BV2 microglia as compared with the cells treated with OGD only (Figure 5). This increase in the expression of TNF-α and IL-1β induced by OGD and exogenous CX3CL1 could be dramatically suppressed by pretreatment of the cells with CX3CR1 siRNA (Figures 5A and 5B) or the p38MAPK inhibitor (20 μmol/L of SB; Figures 5C and 5D). These findings showed that the increased expression of TNF-α and IL-1β induced by the addition of CX3CL1 in OGD-treated BV2 microglia is CX3CR1 and also p38MAPK dependent.

Figure 4.

Figure 4

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Treatment with oxygen–glucose deprivation (OGD) and CX3CL1 significantly increase the expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β in BV2 microglia in vitro. BV2 microglial cells were treated with OGD for 4 hours in the presence of CX3CL1 (0, 12.5, 25, and 50 ng/mL), and then the measurements of TNF-α and IL-1β in the medium were conducted using enzyme-linked immunosorbent assay (ELISA) as described in the Materials and Methods section. (A and B) The levels of TNF-α and IL-1β in the medium of BV2 microglia treated with OGD for 4 hours. (C and D) The contents of TNF-α and IL-1β in the medium of BV2 microglia treated with OGD plus different concentrations of CX3CL1. Data were represented as mean±s.e.m. (n=5). *P<0.05 versus the control (A and B) or 0 ng/mL CX3CL1 (C and D).

Figure 5.

Figure 5

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CX3CR1 shRNA or SB2035080 (SB, a p38MAPK inhibitor) significantly reduce the increased expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β induced by oxygen–glucose deprivation (OGD) and CX3CL1 in BV2 microglia in vitro. BV2 microglial cells were infected with CX3CR1 siRNA or negative siRNA for 72 hours (A and B) or pretreated with 20 μmol/L of SB2035080 only for 1 hour (C and D), then subjected to OGD for 4 hours in the presence of 50 ng/mL of CX3CL1. After the treatments, the measurements of TNF-α and IL-1β were conducted using enzyme-linked immunosorbent assay (ELISA) as described in the Materials and Methods section. (A and C) TNF-α; (B and D) IL-1β. Data were represented as mean±s.e.m. (n=5). *P<0.05 versus the control; #P<0.05 versus OGD; and @P<0.05 versus CX3CL1+OGD.

Bilateral Common Carotid Artery Stenosis and Oxygen–Glucose Deprivation Induced a Significant Increase in CX3CL1 Expression In Vivo and In Vitro

In vivo experiments showed that there was a basal level of CX3CL1 expression in mice brain cortex (Figure 6A—sham), while BCAS could induce a significant increase in CX3CL1 expression in this brain region (Figure 6A). Immunostaining analysis showed that there also was a basal level of CX3CL1 expression in normal cultured neurons (Figure 6B). Meanwhile, OGD/R treatment induced a significant increase in CX3CL1 level in the neurons (Figure 6B) and also in the culture medium (Figure 6C). These findings evidenced that CX3CL1 was not only expressed in neurons but also released from neurons in the brain under the conditions of ischemia.

Figure 6.

Figure 6

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Effects of bilateral common carotid artery stenosis (BCAS) and oxygen–glucose deprivation (OGD) on CX3CL1 expression in the brain cortex in vivo and primary cultured neurons in vitro. Mice were treated with BCAS as described in the Materials and Methods section and then CX3CL1 expression was examined by immunostaining (A). Primary cultured neurons were treated with OGD for 4 hours followed by reoxygenation (R) for 24 hours. The CX3CL1 expression in neurons was then detected by immunostaining (B) and CX3CL1 contents in the neuronal culture medium measured by enzyme-linked immunosorbent assay (ELISA; C). Data were represented as mean±s.e.m. (n=3). *P<0.05 versus the control.

Discussion

In the present study, we showed that ischemia induced by BCAS could lead to white matter lesion, as reflected by the significantly increased white matter rarefaction grading scores and induce cognitive deficits as evidenced by the much longer mean escape latency in ischemic mice. By using CD11b identification, we showed that ischemia induced by BCAS could result in the typical morphologic changes in microglial activation in mice brain in vivo. At the same time, we found that white matter lesions, cognitive deficits, and microglia activation, induced by BCAS, all could be significantly attenuated rather than exacerbated by CX3CR1 siRNA to silence the expression of CX3CR1. These findings indicated that microglial activation induced by ischemia is CX3CR1 mediated and also plays a detrimental rather than protective or beneficial role in the brain of ischemic mice under our experimental conditions.

Once activated, microglia are thought to release a variety of inflammatory and cytotoxic mediators such as TNF-α, IL-1β, and reactive oxygen species contributing to cell damage and cell death.33, 34, 35 We therefore investigated the effects of ischemia on the expression of TNF-α, IL-1β, and IL-6 in mice brain in vivo or by OGD in BV2 microglia in vitro to answer the question of why ischemia induced and CX3CR1-mediated microglial activation plays a detrimental role. We showed that treatment with BCAS or OGD could induce a significant increase in the expression of TNF-α, IL-1β, and IL-6 in mice brain in vivo or in BV2 microglia in vitro, respectively. In addition, we found that the reduced expression of CX3CR1 by CX3CR1 siRNA could lead to a remarkable reduction in the expression of these microglia cytokines in mice brain in vivo or in BV2 microglia in vitro, respectively. The findings implied that the detrimental effect induced by ischemia is because of the significantly increased expression of these microglia cytokines and also evidenced that expression of these cytokines is CX3CR1 mediated in mice brain in vivo or in BV2 microglia in vitro.

To understand the mechanisms involved in the CX3CR1-mediated expression of microglia cytokines, we examined the effects of CX3CR1 siRNA on the expression of CX3CR1 and also p38MAPK, PKC, and microglia cytokines in the brain of BCAS mice in vivo and the effects of CX3CR1 siRNA and SB2035080 on the expression of TNF-α and IL-1β in OGD-treated BV2 microglial cells in vitro. We showed that inhibition of CX3CR1 by CX3CR1 siRNA led to not only a significant reduction in the expression of CX3CR1 but also a remarkable decrease in the contents of p38MAPK, PKC as well as TNF-α, IL-1β, and IL-6 in the brain of BCAS mice in vivo. In in vitro studies, we showed that pretreatment with CX3CR1 siRNA or the p38MAPK inhibitor could significantly attenuate the increased expression of TNF-α and IL-1β induced by OGD in BV2 microglial cells in vitro. These findings plus the consistency in the effects of BCAS on the expression of CX3CR1, p38MAPK, PKC, TNF-α, IL-1β, and IL-6 supported that the expression of p38MAPK and PKC is CX3CR1 dependent and the expression of TNF-α and IL-1β is CX3CR1 and p38MAPK/PKC dependent in the brain of ischemic mice.

CX3CL1 is highly expressed within neurons in the brain.36 It has been reported that ischemia may upregulate CX3CL1 expression by increasing the local concentrations of CX3CL1 production enhancers, including TNF-α and IL-1β.37, 38, 39 In addition, the role of this chemokine in the CX3CR1-associated activation of microglia was well demonstrated.4, 11 These data led us to speculate that ischemia induced by BCAS might be able to promote the expression of CX3CL1 in neurons and the increased expression of CX3CL1 might have a potential role in the development of the detrimental role induced by CX3CR1-mediated microglia activation in the brain of ischemic mice. To test this hypothesis, we investigated the effects of BCAS and OGD/R on CX3CL1 expression in vivo and in vitro and the effects of exogenous CX3CL1 on the expression of TNF-α and IL-1β in OGD-treated BV2 microglial cells in vitro. We showed that CX3CL1 was not only expressed in but also released from neurons and that both BCAS or OGD/R treatment could markedly increase CX3CL1 expression and release in the brain and in cultured neurons. Also, we found that exogenous CX3CL1 had a significant role to increase TNF-α and IL-1β expression in BV2 microglia treated without OGD, and could lead to a further increase in the levels of these proteins in BV2 microglia treated with OGD. More importantly, pretreatment with CX3CR1 siRNA was able to attenuate markedly CX3CL1-induced increase in TNF-α and IL-1β expression in BV2 microglia treated with OGD. Our findings showed that the increased expression of TNF-α and IL-1β induced by CX3CL1 is CX3CR1 mediated and also provide indirect evidence for the involvement of CX3CL1 in CX3CR1- and microglia activation–induced detrimental role in the brain of ischemic mice.

It has been reported that CX3CR1 deficiency or silencing CX3CR1 expression worsens the behavioral impairment and exacerbates the learning and memory deficits in rat model of transient global cerebral ischemic injury4 and that exogenous CX3CL1 reduces ischemia-induced cerebral infarct size and neurologic deficits in the murine model of permanent middle cerebral artery occlusion.19 These suggested that CX3CL1/CX3CR1-mediated microglial activation plays a protective role in ischemic brain rather than a detrimental role as we found in the present study. In addition, we showed that both BCAS and OGD increase CX3CR1 expression in mice brain in vivo and in BV2 microglia in vitro, rather than the decreased expression of the receptor that was found in rats treated with transient global cerebral ischemia reported by Briones et al.4 Moreover, we revealed that exogenous CX3CL1 in vitro and activation of CX3CR1 in vivo both lead to an increase in the contents of TNF-α and IL-1β, however, the opposite findings have been reported.15, 40 Currently, the precise causes of these conflicting observations are unknown and the relevant studies, including microglial physiology, still remain insufficient at present although these conflicting findings might be partly associated with the differences in animal models used, timing of measurement,4 and types of microglia (M1 or M2) activated under differential experimental conditions.20, 41, 42, 43

In summary, we reported that microglial activation induced by ischemia plays a detrimental rather than protective role in the brain of ischemic mice. Based on the in vivo and in vitro findings in the present study, we proposed that ischemia might be able to stimulate the expression of both CX3CL1 in neurons and CX3CR1 in microglia and hence increase the contents of CX3CL1/ CX3CR1 as well as the binding activities of CX3CL1 with CX3CR1 in the brain of the ischemic mice. The increased binding activities, in turn, lead to the activation of microglia as well as the phosphorylation of molecules involved in the signal transduction pathway such as p38MAPK/PKC, and then result in an increase in the generation of TNF-α and IL-1β in microglial cells. The increased harmful cytokines, in the end, led to a detrimental effect in the brain of ischemic mice, and also could promote further increase in the expression of CX3CL144, 45 and induce a progressive cell damage and death in the brain (Figure 7). Collectively, our data provide new insight for the better understanding of the role of the activated microglia and the involvement of CX3CL1/CX3CR1 axis, microglial activation, signal transduction pathway, and microglia cytokines in the development of ischemic injury in the brain, and also implied that CX3CL1/CX3CR1 might be a putative therapeutic target to disrupt the cascade of deleterious events that lead to brain ischemic injury.

Figure 7.

Figure 7

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The speculative mechanism involved in the detrimental effect induced by the CX3CL1/CX3CR1-mediated microglial activation in the brain of ischemic mice. Ischemia (bilateral common carotid artery stenosis, BCAS) might stimulate neurons to express and release CX3CL1 and microglia to express CX3CR1 and hence increase the interactions of CX3CL1 with CX3CR1. This, in turn, activates microglia p38MAPK/PKC signaling pathway and then increase TNF-α and IL-1β production in microglial cells in ischemic mice. The increased harmful cytokines, in the end, led to a detrimental effect in the brain of ischemic mice, and also promote further increase in CX3CL1 expression and induce a progressive cell damage and death in the brain.

The authors declare no conflict of interest.

Footnotes

The studies in our laboratories were supported by the Competitive Earmarked Grants of The Hong Kong Research Grants Council (GRF 466713), National 973 Programs (2011CB510004 and 2014CB541604), General Grant of National Natural Science Foundation of China (NSFC; 81070930,81471108, 31271132 and 31371092), and Key Project Grant of NSFC (31330035-2013).

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