ABSTRACT
Background
Brain injury accompanied by hemorrhage, such as cerebral contusion or intracerebral hemorrhage, leads to the accumulation of activated microglia around the lesion. In addition, microglia at the site of injury can act either damagingly or protectively, depending on the time; for instance, it is damaging in the acute phase and protective in the chronic phase. Moreover, during brain injury, glutamate-induced excitotoxicity leads to secondary damage to neurons. However, the source of glutamate released from cells remains largely unknown. Our previous studies have revealed that cystine/glutamate antiporter system xc- (xc-) in microglia is an important source of glutamate release and that the induction of expression of xCT, a component molecule of xc-, is vital.
Methods
We investigated the effect of microglial xCT on traumatic brain injury using xCT-deficient mice.
Results
In cultured microglia supplemented with crude brain extract and the affected side of the brain injury model accompanied by hemorrhage, the expression level of xCT was increased on the affected side, and induction was observed mainly in microglia. In addition, EAAT2 levels on the affected side decreased. On the affected side, the number of CD80-positive microglia was significantly increased, and the xCT expression rate was elevated in CD80-positive cells. Assuming that xCT in microglia is important, we investigated xCT-deficient mice and microglia-specific xCT knockdown mice and found that the extent of brain damage was milder than in wildtype mice. The proportion of CD80-positive microglia was lower than that in wild-type mice. Assuming that microglial xCT could be a therapeutic target, we performed an experiment using the xCT inhibitor SSZ administered intraperitoneally. The extent of damage was narrowed, and the ratio of CD80-positive microglia was reduced, demonstrating a therapeutic effect.
Conclusion
Thus, microglial xCT is important in the pathology of brain injury accompanied by bleeding and is considered a promising therapeutic target.
Keywords: glutamate, microglia, system xc-, traumatic brain injury, xCT
Brain injury accompanied by hemorrhage is caused by cerebral contusion or intracerebral hemorrhage and is known to cause the accumulation of activated microglia and reactive astrocytes in the surrounding area.1,2,3 In addition, the damaged area and its surroundings are subject to stress caused by blood and its heme content,4 oxidative stress,5 edema,6 and the effects of DAMPs, such as HMGB1 and other leaked components.7, 8
Microglia in damaged areas can act either damagingly or protectively depending on the stage.9 Activated microglia are damaging especially during the acute stage and protective from the chronic stage onward.9 Additionally, in brain injury, excitotoxicity of the nervous system caused by glutamate contributes to secondary damage to the nerves and brain.10, 11 However, the source of extracellular glutamate has remained largely unknown. In addition to releasing inflammatory cytokines, activated microglia have attracted attention as a source of glutamate.12, 13 Our previous reports also revealed that the microglial system xc- (xc-) is an important source of glutamate release and that the induction of the expression of its component molecule, xCT, is vital.13 Xc- forms a heterodimer with 4F2hc on the cell membrane and functions as an antiporter, importing cystine into the cell while exporting glutamate into the extracellular space. In cells expressing the xc- transporter, antioxidants such as glutathione and thioredoxin, which serve as cytoprotective agents, can be produced in abundance.14, 15 However, the glutamate exported by this system can induce excitotoxicity in surrounding neurons.13 We focused on xc- and its component molecule xCT in activated microglia as a potential mechanism of glutamate release, and investigated their roles in the pathology of traumatic brain injury as well as their potential as therapeutic targets.
MATERIALS AND METHODS
Preparation of animals and rearing environment
The C57BL/6 mice were obtained from CLEA Japan (Tokyo, Japan). The xCT knockout mice used in this study were established and reported by H. Sato.16 xCT flox/flox mice and microglia specific knockdown mice xCTflox/flox, Cx3CR1-Cre were purchased from Cyagen Biosciences Inc. (Silicon Valley, CA). Because brain tissue damage, including cerebral hemorrhage, is believed to be more prevalent in men, male mice were used in this study. Wildtype mice (42 mice), xCT knockout mice (20 mice), microglia-specific xCT knockdown mice xCTflox/flox, Cx3CR1-Cre (16 mice), and xCTflox/flox mice (16 mice) (10-14 weeks old) were used in this study. The mice were housed under standardized lighting (06:00-18:00), temperature (25°C), and humidity (approximately 50%), and food and water were provided ad libitum. Experiments were performed using age- and weight-matched animals. The Animal Experiment Committee of Tottori University (Yonago, Japan) approved all procedures (No. 24-Y-24), and all animal experiments were performed in accordance with the relevant guidelines and regulations.
Cell culture
Cultured microglia 6-3 cells were obtained from Wako (Osaka, Japan). The cells were cultured according to the manufacturer’s protocol. Blood components were collected from the heart of C57BL6 mice, and a small amount of distilled water was added for osmotic lysis. Crude extracts of brain tissue equivalent to DAMPs were prepared by intravascular perfusion with saline, followed by the removal of the brain tissue, homogenization of the cerebral cortex with PBS, collection of the soluble fraction by centrifugation, and addition to the cells. LPS (0.5 g/mL) (Sigma-Aldrich, Saint Louis, MO) was added as a positive control. Samples were collected 12 h after the addition and subjected to western blot analysis.
Preparation of traumatic brain injury model
The mice were anesthetized using a mixture of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) (intraperitoneally), and the scalp was immediately incised along the midline to expose the skull. A midline incision was made immediately to expose the skull. At a point 2.5 mm to the right of the middle of the sagittal suture and 2 mm anterior from the lamda level, a 26 G needle was inserted through the skull to a depth of 7 mm into the deep cerebrum to create a brain injury model. The incision was closed, and the anesthesia antagonist atipamezole hydrochloride was administered subcutaneously. Sulfasalazine (SSZ) (Sigma) was used for xCT inhibition experiments. SSZ was dissolved in saline and administered intraperitoneally at 100 mg/kg twice, immediately after needle insertion and 24 hours later. After the procedure, the mice were housed in a 25°C and 60% humidity environment, with food and water available ad libitum. Mouse brains were sampled 24 or 48 hours after needle insertion.
Western blotting and preparations of samples for SDS-PAGE
Brain tissue (24 hours after needle insertion) and cultured microglia were lysed in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 2 mM ethylenediaminetetraacetic acid). The lysates were subjected to SDS-PAGE and transferred to PVDF membranes (Hybond-P; GE Healthcare, Chicago, IL). Membranes were blocked with 3% skim milk/PBS-Tween 0.5% (PBST) solution. Blots were then immersed in PBST solution containing anti-xCT (Abcam, Cambridge, UK), anti-EAAT2 antibody (Abcam), or anti-β-actin (Cell Signaling Technology, Danvers, MA). Immunoreactive signals were detected using HRP-conjugated anti-rabbit IgG or anti-mouse IgG and ECL detection reagents (GE Healthcare) according to the manufacturer’s specifications. The protein content of each sample was measured using a bicinchoninic acid (BCA) protein assay system (Thermo Fisher Scientific, Waltham, MA).
Histological analyses
Hematoxylin and eosin (HE) staining and immunohistochemical analysis were performed using paraffin-embedded sections of mouse brain tissue. Each fixation procedure was performed 48 and 24 hours after needle insertion, respectively. Brains were perfusion-fixed in 4% paraformaldehyde (Wako) and embedded in paraffin. Paraffin sections (5 μm) were deparaffinized by immersing the slides in xylene three times and then rehydrated in graded ethanol solutions. Sections were washed with water and microwaved in 10 mM citrate buffer (pH 6.0). The sections were incubated overnight at 4°C in blocking reagent and reacted with the following primary antibodies: goat anti-GFAP (Abcam), goat anti-Iba-1 (Abcam), and rabbit anti-xCT (Abcam). The sections were rinsed with PBS and incubated with secondary antibodies. For immunofluorescence labeling secondary antibodies, mouse anti-goat IgG-FITC (Santa Cruz) and bovine anti-rabbit IgG-TR (Santa Cruz) were used. Nuclei were counterstained with DAPI. Images were obtained using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). HE staining was performed according to standard protocols. The lesion area was defined as the region surrounding the insertion site where HE staining was reduced, and its width was measured at a point 0.5 mm below the brain surface.
Microglia isolation for flow cytometry
Mouse microglia were isolated 24 after needle insertion using a Percoll density gradient. The mice were perfused with PBS, and their brains were subsequently removed and minced with scissors. Minced brain tissue was digested in a shaking incubator (180 rpm, 37 °C for 30 min) using a digestion solution consisting of RPMI 1640 medium (Gibco, Tokyo, Japan), 0.05% trypsin-EDTA (Gibco), and 4.5 U/mL dispase II (Roche). Digestion was halted by adding an equal volume of RPMI 1640 medium supplemented with 20% fetal bovine serum. The digested tissue was filtered through a 100 μm nylon mesh and centrifuged at 1500 × g for 10 min at room temperature (RT). The pellet was resuspended in 37% Percoll (Sigma) prepared in Hanks’ balanced salt solution (HBSS; Gibco). A three-layer Percoll density gradient was prepared with 70% Percoll at the bottom, 37% in the middle (containing the sample), and 30% on the top, overlaid with HBSS. The gradient was centrifuged at 500 × g for 20 min at 18 °C without braking. Microglia were collected from the interface between the 37% and 70% layers, diluted with HBSS, and centrifuged again at 1,500 × g for 10 min at RT. The final pellet (isolated microglia) was used for flow cytometry.
Flow cytometry analysis
Fc receptors on isolated microglia were blocked using TruStain FcX PLUS (BioLegend, San Diego, CA). The cells were then stained with the following antibodies: PerCP/Cyanine5.5 anti-mouse CD45 (BioLegend), APC/Cyanine7 anti-mouse/human CD11b (BioLegend), PE anti-mouse CD80 (BioLegend), APC anti-mouse CD206 (BioLegend), and anti-xCT (Abcam). DAPI (BioLegend) was used for live/dead staining. After primary staining for 30 min at RT, the cells were washed with PBS and incubated with Alexa Fluor 488 donkey anti-rabbit IgG secondary antibody for 30 min at RT (to detect the xCT antibody). Stained microglia were analyzed using a BD LSR Fortessa X-20 cell analyzer (BD Biosciences), and data were processed using FlowJo software (version 10.10.0; BD Biosciences).
Statistical analyses
Statistical analyses were performed using GraphPad Prism ver 9.5.1 software. The criterion for statistical significance was P < 0.05.
RESULTS
Traumatic brain injury induces xCT expression primarily in activated microglia
In a brain injury model induced by a mild cerebral crush, we hypothesized that blood components and DAMPs released from injured cells contribute to the pathology. To test this, we investigated the induction of xCT expression in cultured microglia following exposure to blood components and crude brain extracts. Both stimuli increased xCT expression levels, comparable to the effect observed with LPS treatment (Fig. 1A).
Fig. 1.
xCT is induced in cultures microglia by TBI-related stimulation. Western blotting using xCT antibody shows induction of xCT by blood and brain lysate as well as LPS stimulation in cultured microglia (12 hours, 6-3 cells).
Similarly, when the expression levels of xCT in brain tissue in a brain injury model were compared between the healthy and affected sides (Fig. 2A), the expression levels of xCT were increased in the affected side. xCT expression was observed mainly in microglia, with only slight induction in astrocytes. Conversely, the expression level of the transporter EAAT2, which is important for glutamate uptake, was decreased in the affected side (Fig. 2B).
Fig. 2.
Traumatic brain injury induces the expression of xCT in the brain. (A) Schematic diagram of TBI model. The point of insertion is demonstrated. (B) The expression of xCT and EAAT2 in the brain. Western blotting using xCT or EAAT2 antibody shows the induction of xCT and the reduction of EAAT2 in the AS of the TBI model mice. (C) Immunostaining of xCT and Iba-1. In AS of the TBI mouse brain, In AS, xCT-positive cells were frequently observed and these cells closely matched with Iba-1-positive cells (white triangle). On the other hand, in CTL, there were few xCT-positive cells, and only a small number of very weakly xCT-positive cells matched with Iba-1-positive cells (open triangle). Scale bar = 100 μm. (D) Immunostaining of xCT and GFAP. In AS of the TBI mouse brain, In AS, most xCT-positive cells were observed as GFAP-negative cells, and a small number of very weakly xCT-positive cells matched with GFAP-positive cells (open triangle). Scale bar = 100 μm. (E–H) Flowcytometry for microglia. (E) CD80-positive microglia (M1 microglia) are significantly abundant in AS compared with CTL side. (mean ± S.D., **P < 0.01, Student t-test) (F) CD80-positive microglia (M2 microglia) are significantly abundant in AS compared with CTL side. n = 10, mean ± S.D., **P < 0.01, Student t-test) (G) xCT-positive microglia are significantly abundant in AS compared with CTL side. mean ± S.D., *P < 0.05, Student t-test. (H) xCT-positive microglia are observed abundantly in M1 microglia in the AS compared with CTL side. (mean ± S.D., *P < 0.05, Student t-test)
Immunohistochemical staining showed that the Iba-1 and xCT signals matched well on the affected side (white triangles). On the healthy side, xCT expression was low, with some Iba-1-positive cells expressing xCT at low levels (open triangles) (Fig. 2C). In contrast, there was little overlap between GFAP-positive and xCT-expressing cells, with only a small proportion showing low expression levels (Fig. 2D).
When the properties of the isolated microglia were examined using flow cytometry, the number of microglia expressing CD80 (Fig. 2E), a marker of M1-type microglia, or CD206, a marker of M2-type microglia (Fig. 2F), increased on the affected side. Furthermore, a significant increase in xCT-expressing microglia was observed on the affected side (Fig. 2G). Detailed analysis revealed that a significantly greater number of xCT-expressing microglia were CD80-positive M1-type. (Fig. 2H)
These findings indicate that brain crush injury activates microglia via blood and DAMPs, inducing xCT expression. Furthermore, the increase in xCT expression and decrease in EAAT2 expression in the affected brain tissue suggest a possible rise in extracellular glutamate levels.
Total xCT deletion mice show reduced lesion extent
To clarify the involvement of xCT in secondary brain injury, we used xCT-deficient mice. The area of cerebral crush injury in xCT-deficient mice was 55.4% smaller than that in wildtype mice (Figs. 3A–C). Flow cytometry showed that the ratio of CD80-positive microglia on the healthy side was not significantly different between xCT KO and wild-type mice, but the ratio on the affected side was lower in xCT KO mice (Fig. 3D). CD206-positive microglia were lower on the healthy side of xCT KO mice compared to wild-type, and the ratio of M2-type microglia was reduced even under normal conditions (Fig. 3E).
Fig. 3.
Genetical deletion of xCT reduces TBI severity. (A)The extent of tissue damage is significantly reduced by xCT deletion. (B) HE staining of the brain tissue from TBI model mice (wildtype). Double arrow demonstrates the width of tissue injury. Scale bar = 500 μm. (C) HE staining of the brain tissue from TBI model mice using xCT-deficient mice. Double arrow demonstrates the width of tissue injury. Scale bar = 500 μm. (D) The relative number of M1 microglia in CTL side and AS from the brain of wildtype or xCT KO mice. The number of CD80-positive (M1) microglia increases in AS significantly. The number of M1 microglia in AS from xCT KO mice is fewer than that from wildtype brain. (mean ± S.D. *P < 0.05, Two-way ANOVA followed by Tukey’s multiple test) (E) The relative number of M2 microglia in CTL side and AS from the brain of wildtype or xCT KO mice. The number of CD206-positive (M2) microglia does not increase in AS from wildtype brain. The number of M2 microglia in CTL side is basically low and significantly increases in AS from xCT KO mice (mean ± S.D., *P < 0.05, Two-way ANOVA followed by Tukey’s multiple test)
Microglia-specific xCT knockdown mice show reduced lesion extent
Similarly, microglia-specific xCT knockdown mice showed 56.1% smaller injury area compared to xCTflox/flox mice (Figs. 4A–C). Flow cytometry showed that the ratio of CD80-positive microglia on the healthy side was not significantly different in microglia-specific xCT KO mice compared to wild-type, but the ratio on the affected side was lower, consistent with findings in whole-body xCT KO mice (Fig. 4D). Regarding CD206-positive microglia, the trend was similar to that in xCT KO mice, though no significant difference was observed (Fig. 4E).
Fig. 4.
Microglia specific knock down of xCT reduces TBI severity. (A) The extent of tissue damage is significantly reduced by microglia specific xCT deletion. (B) HE staining of the brain tissue from TBI model mice (wildtype). Double arrow demonstrates the width of tissue injury. Scale bar = 500 μm. (C) HE staining of the brain tissue from TBI model mice (microglia specific xCT KD). Double arrow demonstrates the width of tissue injury. Scale bar = 500 μm. (D) The relative number of M1 microglia in CTL side and AS from the brain of xCTflox/flox or xCTflox/flox,Cx3CR1-Cre (xCTCX3CR1) mice. The number of CD80-positive (M1) microglia increases in AS both from xCTflox/flox and xCTCX3CR1 mice, but these are not statistically significant. (mean ± S.D., Two-way ANOVA followed by Tukey’s multiple test) (E) The relative number of M2 microglia in CTL side and AS from the brain of xCTflox/flox and xCTCX3CR1 mice. The number of CD206-positive (M2) microglia does not increase in AS from xCTflox/flox mice brain. The number of M2 microglia in CTL side is basically low in CTL side. (mean ± S.D., *P < 0.05, Two- way ANOVA followed by Tukey’s multiple test)
Inhibition of xCT improves lesion extent
Based on these results, we hypothesized that microglial xCT could be a therapeutic target in the brain injury model and performed a treatment experiment using an xCT inhibitor. SSZ is a broadly acting immunosuppressant used for ulcerative colitis and rheumatoid arthritis and is known to inhibit xCT.17, 18 Because oral administration leads to its breakdown by intestinal bacteria into 5-ASA and fapyridine, SSZ was administered intraperitoneally in this experiment. Although the half-life of SSZ is short (approximately 12 hours), it was administered only twice—on the day of injury and one day later—assuming short-term treatment in the acute phase. Forty eight hours later, the SSZ-treated group showed a significantly smaller injury area (43.9% smaller) compared to the saline-treated group (Figs. 5A–C). Flow cytometry showed that the ratio of CD80-positive microglia on the CTL side was not significantly different between sham-treated and SSZ-treated mice, but the ratio on the affected side was lower in SSZ-treated mice (Fig. 5D). CD206-positive microglia were lower on the CTL side of SSZ-treated mice compared to sham-treated mice, and the ratio of M2-type microglia showed tendancy to reduced even under normal conditions, but was not statistically significant (Fig. 5E).
Fig. 5.
Inhibition of xCT by sulfasalazine reduces severity of TBI. (A) The extent of tissue damage is significantly reduced by sulfasalazine (SSZ) treatment. (B) HE staining of the brain tissue from TBI model mice (wildtype-saline, i.p.). Double arrow demonstrates the width of tissue injury. Scale bar = 500 μm. (C) HE staining of the brain tissue from TBI model mice (wildtype-SSZ, i.p.). Double arrow demonstrates the width of tissue injury. Scale bar = 500 μm. (D) The relative number of M1 microglia in CTL side and AS from the brain of sham treatment or SSZ-treated mice. The number of CD80-positive (M1) microglia increases in AS significantly. The number of M1 microglia in AS from SSZ-treated mice is fewer than that from wildtype brain. (mean ± S.D., *P < 0.05, Two-way ANOVA followed by Tukey’s multiple test)(E) The relative number of M2 microglia in CTL side and AS from the brain of saline treatment or SSZ-treated mice. The number of CD206-positive (M2) microglia does not increase in AS both saline or SSZ-treated mice brain. (mean ± S.D., *P < 0.05, Two-way ANOVA followed by Tukey’s multiple test)
These findings suggest that acute-phase xCT inhibition can reduce damage in traumatic brain injury and that xCT is a promising therapeutic target.
DISCUSSION
The role of extracellular glutamate in secondary brain tissue damage from various causes has drawn increasing attention.10, 11 Extracellular glutamate is implicated in delayed neuronal death after transient ischemia,19 tissue damage and edema following cerebral hemorrhage,20 neuronal death after seizures,21 and neuronal loss in neurodegenerative diseases.22 However, the source of extracellular glutamate in these conditions remains unclear. Recent reports of microglial activation in these pathologies led us to investigate the microglial xc- as a potential source of extracellular glutamate.13
xc- is known to transport cystine, an oxidized dimer of cysteine, into cells. This is crucial under oxidative stress, as cystine supports the production of the antioxidant glutathione (GSH), which contains a cysteine residue.14, 15 Therefore, deletion or inhibition of xCT may suppress pathological microglial activation around the injury site. To explore this, we examined the effect on the balance between two functionally distinct microglial phenotypes: classical “injury-type” (M1) and “protective-type” (M2).23 Under normal conditions, xCT expression is extremely low-barely detectable in microglia and astrocytes, and absent in neurons. However, in various brain injury models, its expression is upregulated in microglia and astrocytes.13 In our traumatic brain injury model, xCT expression was markedly increased in microglia around the lesion site on the affected side (Fig. 2).
The ratio of M1 cells was increased in brain tissue from the injured side of wildtype mice 24 hours after brain injury compared to the healthy side. However, this increase was attenuated in xCT knockout mice, with the M1 cell ratio significantly lower than that of wildtype mice. Activated M1 microglia are known to produce neurotoxic cytokines such as TNF-α and IL-1β.13 In our flow cytometry analysis, the proportion of xCT-expressing cells was also elevated (Fig. 2H). The increase in CD80-positive microglia, along with reduced EAAT2 expression, critical for glutamate uptake, on the injured side, suggests a rise in extracellular glutamate levels.
To demonstrate that microglial, rather than astrocytic, xCT is critical in this pathological context, we employed microglia-specific xCT knock down mice in addition to whole-body xCT knock out mice (Fig. 4). The findings in microglia-specific xCT knockdown mice largely mirrored those observed in whole-body xCT deficient mice and in SSZ-treated animals, reinforcing the conclusion that microglial xCT plays a central role in the pathogenesis of brain injury in this model (Fig. 5).
We have previously demonstrated using in vivo microdialysis that glutamate released via microglial xCT contributes to depression-like behavior and cognitive impairment in conditions involving microglial activation, such as post-sepsis.13 Although direct glutamate measurements were not performed in this study, nor were experiments conducted with NMDA or AMPA receptor antagonists, our previous findings suggest that a similar mechanism may underlie the current observations.13 As extracellular glutamate induces excitotoxicity, neuronal death, and behavioral dysfunction—including cognitive deficits and depressive symptoms—the source of glutamate release, such as xCT, warrants attention as a potential therapeutic target.
Our results suggest that the microglial xc-/xCT is a viable therapeutic target for traumatic brain injury (Fig. 6). SSZ, a widely used immunosuppressant for conditions such as ulcerative colitis and rheumatoid arthritis, has been shown to inhibit xCT. Notably, SSZ can cross the blood-brain barrier via the Oatp2b1 transporter. Therefore, SSZ may reduce secondary brain damage by suppressing extracellular glutamate derived from microglial xCT following injury. Moreover, given that SSZ’s therapeutic effects were observed with short-term administration in the acute phase, and considering xCT expression is minimal in healthy brain tissue, the risk of adverse effects is likely low. These findings support xCT inhibition as a promising therapeutic approach for managing hemorrhagic brain injury.
Fig. 6.
Microglial xc-/xCT is a viable therapeutic target for traumatic brain injury. The expression level of xCT in microglia under normal condition is low (left). TBI induces xCT expression in microglia, and causes glutamate release and following excitotoxicity (middle). xCT inhibitor SSZ prevents glutamate release and reduces excitotoxicity (right).
Acknowledgments
Acknowledgments: Preparation of the brain tissue sections was supported by the Technical Department at Tottori University. We thank Ms Kanon Sato for assistance with western blotting. This work was supported by JSPS KAKENHI Grant Number 20H03577, 25K02572, and 24K10643. We would like to thank Editage (www.editage.jp) for English language editing.
Footnotes
The authors declare no conflict of interest.
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