Adult male C57BL/6 mice (8–10 weeks, 23–25 g) were purchased from the Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animal experiments were approved by the Institutional Animal Care and Use Committee of Capital Medical University. Food and water were available ad libitum. All efforts were made to minimize animal suffering and the number of animals used. Animals were randomly divided into: (1) sham; (2) stroke plus vehicle; and (3) stroke plus curcumin groups. In this study, a total of 80 mice were used for infarct volume measurements (23 mice), behavioral tests and immunohistochemical examinations (42 mice), and mRNA or protein expression measurements (15 mice).

Anesthesia was induced with 2% isoflurane in 70% nitrogen/30% oxygen gas mixture. Rectal temperature was maintained at 37 ± 0.5°C with a heating pad. A ~2 cm incision was made between the right eye and ear. Using a surgical microscope, the temporal muscle was dissected to expose right zygomatic arches and squamosal bone. A craniotomy was performed and the right MCA was occluded distal to the lenticulostriate branches with bipolar electrocautery (Goldbov Photoelectronics CO. Ltd, Wuhan, China). The middle cerebral artery occlusion (MCAO) was accompanied by 15-min bilateral occlusion of the common carotid artery (CCA). Regional cerebral blood flow (rCBF) was monitored with a two-dimensional laser speckle imager (Perimed AB, Järfälla, Sweden). Mice with rCBF reduction less than 30% of baseline levels were excluded from further experiments. Sham-operated mice were manipulated in the same way, but the MCA and CCA were not occluded.

Curcumin (Sigma-Aldrich) was dissolved in 5 mol/L NaOH, titrated to pH 7.4 using 1 mol/L HCl, and then diluted with saline. Ischemic mice were subjected to intraperitoneal injections of 150 mg/kg curcumin or the same volume of vehicle. Injections were performed 0 h and 24 h after reperfusion of the CCA.

At 72 h after cerebral ischemia, mice were decapitated and brains were rapidly removed on ice. Brains were sliced into 1 mm-thick coronal sections and stained with 2% 2, 3, 5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich), as previously described (Liu X. et al., 2016). The infarct area was calculated as the area of the contralateral hemisphere minus the noninfarcted area of the ipsilateral hemisphere by a person blinded to the experiment group. Infarct volumes were determined using National institutes of Health ImageJ software. The total infarct areas were multiplied by the thickness of the brain sections to obtain the infarct volumes.

Sensorimotor functional recovery after stroke was measured before or 3, 5, 7 and 10 days after stroke with the adhesive removal test, as described in prior studies (Wang et al., 2017). In brief, a mouse was placed in a cage for 1 min. An adhesive tape (50 mm²) was applied to the distal radial region of the right forelimb as a tactile stimulus. The time to contact and the time to remove the tape were both recorded. Each animal was tested three times with a cutoff time of 120 s per trial. The data are presented as the mean time to contact and the mean time to remove the tape on each testing day. The investigators performing the assessments were blinded to experimental group assignments.

The Modified Garcia Score system was used to assess sensorimotor functions at 3, 5, 7 and 10 days after dMCAO by an observer blinded to the experimental groups. Five tests were performed: body proprioception, vibrissae touch, limb symmetry, lateral turning and forelimb walking with scores of 0–3 for each test, as previously reported studies (Wang et al., 2017). The data from pre-stroke tests were defined as the baseline.

Brain slices were prepared and subjected to immunohistochemistry, as previously published (Pan et al., 2015). Mice were perfused with saline and 4% paraformaldehyde. Brains were then removed, followed by cryoprotection in 30% sucrose. The brains were sliced into 20 μm-thick coronal sections at −25°C using a freezing microtome (CM3050S, Leica, German). The slices were then subjected to immunofluorescent staining. Non-specific staining was blocked with 10% normal serum. Next, brain slices were incubated with mouse anti-CD16 (1:100, BD Pharmingen, San Jose, CA, USA), goat anti-CD206 (1:100, R&D Systems, Minneapolis, MN, USA), and rabbit anti-Iba-1 (1:100, Wako, Richmond, VA, USA) overnight at 4°C. The sections were then treated with fluorophore-conjugated secondary antibodies (Invitrogen Corporation, Carlsbad, CA, USA)—goat anti-rabbit antibody conjugated to Alexa 594 (1:400), goat anti-mouse antibody conjugated to Alexa 488 (1:400), and donkey anti-goat antibody conjugated to Alexa 488 (1:400), at room temperature for 2 h. All images were captured with a fluorescence microscope (DM4000 B LED, Leica Microsystem, Germany) and analyzed by a blinded observer with ImageJ. Cell numbers were calculated from three randomly-selected microscopic fields, and three consecutive sections were analyzed for each brain.

The BV2 microglia cell line (Cell Center, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing, China) was cultured in RPMI 1640 (Cat No.: C11875500BT, Gibco), supplemented with 10% fetal bovine serum (Cat No.: 16000-044, Gibco) and 1% penicillin-streptomycin (Cat No.: 15070-063, Gibco). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and the medium was changed every 2 days. For M1 stimulation, lipopolysaccharide (LPS, 100 ng/mL) and IFN-γ (20 ng/mL) were added to BV2 microglia cultures. Different concentrations of curcumin were added to the cultures immediately following the application of M1 inducers. After 48 h of treatment, cells were collected for RT-PCR and the supernatant was harvested for the detection of cytokines by ELISA.

The cytotoxicity of curcumin against BV2 microglia cell line was analyzed by a lactate dehydrogenase (LDH) cytotoxicity assay kit (Cat No.: C0017, Beyotime, Shanghai, China) according to the manufacturer’s protocol. Absorbance was measured at 490 nm by a microplate reader (Cat No.: Synergy HT, Biotek, Winooski, VT, USA). All cell culture experiments were performed in duplicate and repeated six times.

Total RNA was extracted from the cortex of C57BL/6 mice or from the BV2 microglia cell line (Cell Center, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing, China) by RNeasy® Lipid Tissue Mini Kit (Cat No.: 74804, QIAGEN) according to manufacturer’s instructions. Three microgram RNA were reverse-transcribed into cDNA using the SuperScript^™ III First-Strand Synthesis SuperMix for qRT-PCR (Cat No.: 11752-250, Invitrogen). RT-PCR was performed using quantitative PCR systems (Applied Biosystems® 7500 Real-Time PCR Systems, Thermo Fisher Scientific, Waltham, MA, USA) with corresponding primers (Table 1, Invitrogen) and a fluorescent dye (RT2 SYBR® Green FAST Mastermixes, Cat No.: 330603, QIAGEN). The cycle time (CT) was normalized to GAPDH in the same sample. The expression levels of mRNAs were reported as fold changes vs. sham control.

Cytokine production (IL-1β, IL-2, IL-12p70, IL-13 and TNF-α) in the BV2 culture media was determined by ELISA (Cusabio Co., Ltd., Wuhan, China), according to the manufacturer’s instructions. Each sample was assayed in triplicate.

Power analyses were used to determine the number of mice within each experimental group, according to our past experience with similar measurements (α = 0.05 and β = 0.20). All data are presented as mean ± SEM. The Student’s two tailed t-test was used for comparisons of two experimental groups. Differences in means across multiple groups were analyzed using one-way or two-way ANOVA, depending on the number of independent variables. Differences in means across multiple groups with multiple measurements over time were analyzed using two-way repeated measures ANOVA. When the ANOVA revealed significant differences, the Bonferroni post hoc test was used for pairwise comparisons between means.

Curcumin (150 mg/kg) was administered intraperitoneally immediately after dMCAO and 24 h after surgery (Figure 1A). As shown in Figures 1B–D, curcumin treatment significantly reduced infarct volumes compared to vehicle. rCBF was measured before and after dMCAO with a two-dimensional laser speckle imager (Figure 1E). For all animals assigned to curcumin or vehicle groups, the rCBF after dMCAO was reduced to about 30% of pre-ischemic baseline values (Figure 1E). There was no statistical difference in rCBF reduction between curcumin and vehicle-treated groups (Figure 1F), verifying that outcome differences between the experimental and control groups cannot be attributed to different degrees of the original ischemic injury.

To evaluate the effects of curcumin treatment on functional outcomes after dMCAO, sensorimotor deficits were measured by the adhesive removal test and modified Garcia score system at 3, 5, 7 and 10 days post-stroke. Curcumin treatment significantly improved neurological performance in the adhesive removal test after dMCAO, as manifested by a consistent reduction in the times to contact and then remove the tape from the compromised limb (Figures 2A,B, respectively). In addition, sensorimotor functions such as body proprioception (Figure 2C), limb symmetry (Figure 2E), and lateral turning (Figure 2F) were all significantly improved in curcumin-treated mice compared to vehicle-treated mice, resulting in an increase in the total neurological score, especially at day 3–5 after stroke (Figure 2H). There was no significant difference in vibrissae touch (Figure 2D) or forelimb walking (Figure 2G) between vehicle and curcumin-treated groups. These data reveal improvements in some aspects of neurological function at early timepoints after stroke with curcumin.

Polarization of microglia plays a critical role in the pathological progression of ischemic stroke (Hu et al., 2012). Thus, the effect of curcumin on microglial polarization was examined by RT-PCR. RNA samples were prepared from dMCAO mice treated with curcumin or vehicle. As shown in Figure 3, the expression of M1 markers (TNF-α, IL-12, CD16, CD32 and iNOS) and M2 markers (Arg-1 and YM1/2) were all significantly increased in vehicle-treated stroke mice 3 days after dMCAO. Curcumin treatment significantly inhibited this increase of M1 markers and enhanced the expression of M2 markers 3 days after dMCAO (Figure 2).

To further assess whether curcumin regulates microglial polarization in the brain after dMCAO, brain sections were double-stained for Iba-1 (microglial marker) and CD16 (M1 marker) or CD206 (M2 marker). As shown in Figures 4A,C, the percentage of CD16⁺Iba-1⁺ cells among total Iba-1⁺ microglia/macrophages was significantly higher in vehicle-treated mice compared to curcumin-treated stroke mice. Moreover, curcumin administration significantly increased the percentage of CD206⁺Iba-1⁺ M2 microglia/macrophages after dMCAO (Figures 4B,D). Taken together, these results demonstrate that curcumin treatment promotes M2 polarization and inhibits M1 polarization after stroke, consistent with its neuroprotective properties.

The BV2 microglial cell line was used to further determine the effects of curcumin on microglial polarization. First, we performed the LDH assay to determine curcumin cytotoxicity in BV2 microglia cells (Figure 5A). Treatment with 6.25, 12.5 and 25 μmmol/L curcumin showed no significant effects on microglial survival. Treatment with 35 μmmol/L curcumin exerted a significantly cytotoxic effect on BV2 microglia cells. Therefore, the 12.5 and 25 μmol/L concentrations were selected for further experiments.

BV2 microglial cells were treated with LPS (100 ng/mL) and IFN-γ (20 ng/mL) for 48 h to induce the M1 phenotype. As shown in Figures 5B–E, the mRNA expression of M1 markers (TNF-α, IL-12, CD16 and CD32) was increased significantly in BV2 microglia after stimulation with LPS and IFN-γ, but markedly reduced after treatment with 12.5 or 25 μmmol/L curcumin. However, the mRNA expression of iNOS (M1 marker, Figure 5F) was drastically upregulated by LPS and IFN-γ and not significantly reduced with curcumin. In contrast to the upregulation of M1 markers with LPS and IFN-γ, the mRNA expression of M2 markers (CD206 and Arg-1) was reduced by these two pro-inflammatory stimuli. The 12.5 or 25 μmmol/L concentrations of curcumin partially reversed this reduction of CD206 expression (Figure 5G), but showed only marginal effects on Arg-1 expression (Figure 5H). We conclude that most, but not all, M1 markers are almost completely inhibited by curcumin treatment in stimulated microglial cells, whereas M2 markers are partially upregulated.

Next, we performed ELISAs to measure the release of pro-inflammatory cytokines in LPS + IFN-γ-stimulated BV2 cells. LPS and IFN-γ dramatically increased the production of pro-inflammatory cytokines from BV2 cells, including TNF-α, IL-12p70, IL-6, IL-2 and IL-1β (Figure 6). Curcumin treatment significantly reduced the production of pro-inflammatory cytokines including TNF-α, IL-12p70 and IL-6. However, the secretion of IL-1β and IL-2 was not dramatically attenuated with curcumin administration at 12.5 or 25 μmmol/L. Taken together, our in vitro data demonstrate that curcumin promotes M2 polarization and inhibits M1 polarization of microglia, and inhibits production of pro-inflammatory cytokines from M1-stimulated microglia. Collectively, these findings suggest a titration of polarization status by curcumin, both in vivo and in vitro.