Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke

Background Ischemic stroke is a leading cause of disability worldwide and characteristically accompanied by downregulation of the Wnt/β-catenin signaling. Activation of Wnt/β-catenin signaling emerges to attenuate neuroinflammation after ischemic stroke; however, its effect on modulating microglial polarization is largely unknown. Here, we explored whether Wnt/β-catenin pathway activator TWS119 facilitated long-term neurological recovery via modulating microglia polarization after experimental stroke. Methods Ischemic stroke mice model was induced by permanent distal middle cerebral artery occlusion plus 1 h hypoxia. TWS119 was administrated from day 1 to 14 after stroke. Neurological deficits were monitored up to 21 days after stroke. Angiogenesis, neural plasticity, microglial polarization, and microglia-associated inflammatory cytokines were detected in the peri-infarct cortex at days 14 and 21 after stroke. Primary microglia and mouse brain microvascular endothelial cell lines were employed to explore the underlying mechanism in vitro. Results TWS119 mitigated neurological deficits at days 14 and 21 after experimental stroke, paralleled by acceleration on angiogenesis and neural plasticity in the peri-infarct cortex. Mechanistically, cerebral ischemia induced production of microglia-associated proinflammatory cytokines and priming of activated microglia toward pro-inflammatory polarization, whereas TWS119 ameliorated microglia-mediated neuroinflammatory status following ischemic stroke and promoted angiogenesis by modulating microglia to anti-inflammatory phenotype. The beneficial efficacy of TWS119 in microglial polarization was largely reversed by selective Wnt/β-catenin pathway blockade in vitro, suggesting that TWS119-enabled pro-inflammatory to anti-inflammatory phenotype switch of microglia was possibly mediated by Wnt/β-catenin signaling. Conclusions Wnt/β-catenin pathway activator TWS119 ameliorated neuroinflammatory microenvironment following chronic cerebral ischemia via modulating microglia towards anti-inflammatory phenotype, and facilitates neurological recovery in an anti-inflammatory phenotype polarization-dependent manner. Activation of Wnt/β-catenin pathway following ischemic stroke might be a potential restorative strategy targeting microglia-mediated neuroinflammation.


Background
Ischemic stroke is a leading cause of disability worldwide [1]. Hyperacute intravenous thrombolysis and endovascular intervention have been performed in clinical practice. Nevertheless, the low revascularization rates due to short time window and side effects frequently cause cerebral ischemic injury [2]. In response to cerebral ischemia, the brain initiates self-repair mechanisms and undergoes a post-stroke neurorestorative process. Angiogenesis and neural plasticity are major post-stroke neurorestorative elements. Newborn cerebral microvessels derived from angiogenesis have been clinically regarded as the III level collateral circulation for restoring blood supply to the ischemic brain [3]. Extensive rewiring of remaining neuronal connections during the neural plasticity process leads to post-stroke cortical map rearrangement and functional improvement [4]. Unfortunately, the neurorestorative potential of the adult brain is very limited; ischemic stroke patients often suffer long-term neurological deficits [5]. Seeking promising strategies on facilitating post-stroke angiogenesis and neural plasticity is paramount for long-term functional improvement.
Post-stroke angiogenesis and neural plasticity are accompanied by long-lasting neuroinflammation, which provides a promising therapeutic opportunity to improve neurological outcome [6]. Microglia, as the main resident immune cells, display a beneficial efficacy in ameliorating inflammatory microenvironment for neurovascular remolding via shifting polarization from pro-inflammatory phenotype to antiinflammatory phenotype [7,8]. Indeed, mounting evidences demonstrate that the microglia with anti-inflammatory phenotype plays a neurorestorative role during the recovery period of ischemic stroke through secreting antiinflammatory cytokines [9,10]. Anti-inflammatory polarized microglia is a novel therapeutic target for post-stroke neurological recovery [7,8,11].
The Wnt canonical or Wnt/β-catenin pathway is a key mechanism involved in the maintenance of neuronal homeostasis [12]. Deregulation of the Wnt/β-catenin pathway contributes to the pathological progression following neurodegenerative diseases, and it is emerging that the Wnt/β-catenin pathway is involved in neuroinflammation following cerebral ischemia [13]. Growing evidences suggest that activation of the Wnt/β-catenin signaling pathway modulates microglial polarization in vitro [14,15] Interestingly, activation of the Wnt/βcatenin pathway inhibits the release of proinflammatory cytokines and promotes switch of microglial polarization from pro-inflammatory phenotype to anti-inflammatory phenotype in hemorrhagic stroke model [16]. However, the exhaustive effect of Wnt/β-catenin pathway on microglial polarization following ischemic stroke is largely unknown. 4,6-disubstituted pyrrolopyrimidine (TWS119), a specific inhibitor of glycogen synthase kinase (GSK)-3β, increases the protein level of β-catenin and activate the Wnt signaling pathway in the ischemic brai n [17]. Furthermore, the Wnt canonical pathway activator TWS119 exerts favorable effects on immuneinflammation modulation [18,19] and blood-brain barrier protection [17]. We speculated that TWS119 might show activated microglia toward anti-inflammatory phenotype and further facilitated neurological recovery following ischemic stroke. In the current study, we sought to examine the efficacy of the Wnt canonical pathway activator TWS119 on modulating microglial polarization in a focal cerebral ischemia model and focused on whether TWS119 facilitated neurological recovery in an antiinflammatory phenotype polarization-dependent manner.

Animals
Adult male C57BL/6 mice (weight 23-25 g, 8-10 weeks) of SPF grade were obtained from the Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). Mice were housed in a home cage with controlled temperature (22 ± 3°C) and humidity (60% ± 5%) under a 12-h light/ dark cycle and were allowed water and food ad libitum.
Ischemic stroke was induced in male mice (10 weeks old) by permanent distal middle cerebral artery (MCA) occlusion plus 1 h hypoxia [20]. The operation was performed in a sterile operating room. Anesthesia was induced with isoflurane (induction dosage 3%; maintenance dosage 1.5%) in air mixture. During anesthesia, artificial tear ointment was applied for eye protection and lubrication, and sucking was performed regularly to prevent airway obstruction. The right MCA was permanently coagulated distal to the lenticulostriate branches with lowheat bipolar electrocautery (Bovie Aaron Medical, USA). To increase lesion size and consequently behavioral deficits, mice were immediately placed in a hypoxia chamber containing 8% oxygen for 1 h after MCA occlusion. Rectal temperature of each mouse was maintained at 37 ± 0.5°C during the molding process using a heating pad. Mice were excluded from the study with the following conditions: subarachnoid hemorrhage occurred during operation; a recanalization of the MCA after two attempts of electrocoagulation; regional cortical cerebral blood flow reduction less than 30% of the baseline level. Shamoperated mice were subjected to the same surgery and hypoxia, but not occlude MCA.
TWS119 (diluted in 1% DMSO, Selleck, Houston, TX, USA) was administrated to mice by intraperitoneal injection once daily from days 1 to 14 after surgery according to a treatment strategy described previously [7]. Intraperitoneal injection followed the aseptic principle and recommended procedures to avoid complications, such as peritonitis. The dose (10 mg/kg) of TWS119 was selected based on our previous results in the supplementary material. Mice were divided into 3 groups following the principle of randomization (random number): Sham group: sham-operated mice; Vehicle group: mice were subjected to cerebral ischemic injury and vehicle (1% DMSO) treatment; TWS119 group: mice were subjected to cerebral ischemic injury and TWS119 treatment. The experimental scheme is schematically shown in Fig. 1a. Brain tissue for protein, gene, and cytokine detection were harvested from the peri-infarct region as described previously [21]. Immunofluorescence images were collected from the region of interest (ROI) in the ipsilateral peri-infarct cortex. The peri-infarct region (0.5-mm-wide cortical region around brain infarction) and ROI are schematically shown in Fig. 1b.

Primary microglia
Primary microglia cells were prepared from the cerebral cortex of neonatal C57BL/6 mice at P1-P2 as previously reported with some modifications [15]. Briefly, mixed glial cells were cultured glucose DMEM (Cat No.: 11965-084, Gibco) supplemented with 10% fetal bovine serum (Cat No.: 16000-044, Gibco) and 1% penicillinstreptomycin (Cat No.: 15070-063, Gibco) and incubated at 37°C in a humidified incubator containing 5% CO 2 and 95% air (normoxic conditions) for 10 days. Next, microglia cells were separated from the mixed glia cells by shaking the flasks at 200 r.p.m at 37°C for 2 h and collected by centrifugation. Finally, the microglia cells were plated into six-well plates (1 × 10 6 /well) with fresh medium for 24 h before ready for further treatment. The primary microglia cells were determined by immunofluorescence using ionized calcium-binding adapter molecule 1 (Iba-1, 1:500, Wako). The eligible purity of the obtained microglia cells was more than 95%.
For pro-inflammatory phenotype polarization, serumstarved microglia were stimulated with lipopolysaccharide (LPS, 100 ng/ml, Sigma) plus interferon-γ (IFN-γ, 20 ng/ml, Sigma) for 24 h in normoxic conditions [22]. TWS119 (10 μM) and/or the reversible inhibitor of Wnt/β-catenin signaling pathway (IWR-1, 10 μM, Calbiochem) were used for treatment in vitro and added to the microglia cultures immediately following the application of LPS plus IFN-γ. IWR-1, a tankyrase inhibitor targeting Axin, lead to a degradation of β-catenin. The microglia cells were divided into 5 groups: control: microglia cells without LPS plus IFN-γ stimulation and any treatment; LPS+IFN-γ: microglia cells stimulated Fig. 1 Experimental outline and schematic diagram of brain section. a Experimental outline: TWS119 or BrdU were administrated intraperitoneally once daily from day 1 to 14 or from day 7 to 14 after stroke. Neurobehavioral tests were performed at days 1, 7, 14, and 21 after stroke. Angiogenesis, neural plasticity, microglial polarization and inflammatory cytokines were detected at the indicated time point. Histological assessment was assessed at day 21. The number of mice in each group for each test were shown in parentheses. b Schematic diagram of brain section. Green squares indicated the region of interest in the ipsilateral peri-infarct cortex, in which immunofluorescence images were collected. Yellow strip (0.5 mm wide) indicated peri-infarct region, in which brain samples for qRT-PCR and ELISA were harvested. stroke, focal cerebral ischemia; IF, immunofluorescence; qRT-PCR, quantitative real time polymerase chain reaction; ELISA, enzyme linked immunosorbent assay with LPS plus IFN-γ; LPS+IFN-γ+TWS119: microglia  cells stimulated with LPS plus IFN-γ and treated with  TWS119; LPS+IFN-γ+IWR-1: microglia cells stimulated  with LPS plus IFN-γ and treated with IWR-1; LPS+IFN-γ+TWS119+IWR-1: microglia cells stimulated with LPS plus IFN-γ and treated with TWS119 plus IWR-1. After 24 h of incubation, the β-catenin and pro-inflammatory/ anti-inflammatory phenotype marker of microglia cells were detected; microglia supernatants were collected and used as conditioned media (CM); the inflammatory cytokines in microglia CM were evaluated.

Mouse brain microvascular endothelial cell line
The mouse brain microvascular endothelial cell line bEnd.3 (American Type Culture Collection (ATCC), Manassas, VA, USA) were used to evaluate angiogenesis in vitro.
Oxygen-glucose deprivation (OGD) injuries were used to mimic post-stroke ischemic damage as previously described [23]. In brief, bEnd.3 cells were cultured with glucose-free DMEM and incubated for 6 h in a humidified incubator containing 5% CO 2 and 95% N 2 at 37°C.
Pro-angiogenic structures of OGD bEnd.3 cells in DMEM and CM (microglia supernatant) were detected by tube formation assay [8]. Firstly, the bEnd.

Neurobehavioral test
CatWalk test, Adhesive Removal test, Accelerated Rotarod test, and modified neurological severity scores (mNSS) were used for assessing neurological deficits. CatWalk test, Adhesive Removal test, and Accelerated Rotarod test were performed three times for each mouse and needed 5 days of training for adaptability.
CatWalkXT (v10.6, Noldus Information Technology) test was used to measure Walking performance of ischemic mice as described previously [24]. Each mouse walked freely through a corridor on a glass walkway illuminated with beams of light from below. Walking at a steady speed (no stopping, rearing, or grooming) was defined as a successful walking trial, which was performed 3 times for each mouse. The footprints were collected using a camera and then analyzed using CatWalkXT software. Swing speed (cm/s), stride length (cm), and paw intensity (arbitrary units, 0-255) of the left front limb (cerebral ischemia-affected limb) were selected for gait analysis.
Adhesion Removal test was selected to monitor sensorimotor function and postural asymmetry ischemic mice [25,26]. Briefly, two adhesive tapes (0.3 × 0.4 cm 2 ) were attached to each paw of the mice with equal pressure; the order of placing adhesive (right or left) was distinct between each animal and each session. The mice were placed in a Perspex box; contact time (the point that the mouse reacts to the presence of adhesive strips) and Adhesive Removal time were collected, with a maximum of 120 s. Accelerated Rotarod (Ugo Basile 47600, Ugo-Basile, Biological Research Apparatus, Milan, Italy) test was performed to evaluate motor coordination and learning function of ischemic mice according to a previous study [27]. The mice were placed on a gradually accelerated rotating rod (4 to 10 rpm, 5 min). Resident time on the rotating rod (latency time) of each mouse was recorded.
Modified neurological severity scores (mNSS) were performed to evaluate functional impairments [28]. The scores were graded on a scale ranging from 0 to 18 (normal score 0, maximal deficit score 18); the higher the scores, the worse neurological function was. The scores for each mouse were recorded at days1, 7, 14, and 21 after ischemic injury.

Histological assessment
Cortical width index (CWI) was used to assess histological damage as described previously [7]. Whole brain images were captured using a digital camera and analyzed using the ImageJ software (NIH, USA). The injury contralateral width (W.contra) from the lateral edge to the midline and the injury ipsilateral width (W.ipsi) from the edge of cortical cavitation to the midline were measured. Measurements were performed at the midpoint of the forebrain. CWI = W.ipsi × 100%/W.contra.
The number and intensity of Synaptophysin + , PSD95 + , GAP43 + , and SMI312 + puncta were acquired using a confocal laser scanning microscope (Zeiss LSM880, German) and analyzed with MetaMorph software (Molecular Devices, v. 2.8.5) as described previously [4]. CD31positive blood vessels were highlighted using the ImageJ software; the area of CD31 positive blood vessels were automatically calculated by the software and expressed as the object area fraction of ROI [29]. Quantification of CD31-positive vessels was carried out by counting them in ROI as described previously [7]. The density of CD31positive vessels is expressed as the number of positive cells per mm 2 . Determination of the percentage of colabeled CD206 and Iba1 cells was achieved by counting the Iba1 + and co-labeled cells within the ROI using the ImageJ software. The percentage of co-labeled cells was calculated as: (100% × Total number of double positive cells/Total number of Iba1 + cells [29]. The percentage of co-labeled CD16/32 and Iba1 cells and co-labeled BrdU and CD31 cells were also determined in the same way. For each immunohistochemical staining item analysis, three sections were quantified and averaged per mouse, and each section contained three regions of interest in the peri-ischemic region.

Enzyme-linked immunosorbent assay and Griess reaction
Brains were harvested on days 14 or 21 after ischemic stroke and sectioned into seven 1-mm-thick coronal sections. Slices containing infarct were selected and then frozen on dry ice. For slices on day 14, the cortex tissues (about 15 mg per mouse) were taken from the periinfract region; for slices on day 21, the cortex tissues were taken at the identical anatomical location as the slices on day 14. Specimens were homogenized in 450 μl of extraction buffer per 15 mg tissue wet weight. The tissue homogenates were centrifuged at 4°C for 20 min to remove the cellular debris, and then the supernatants were collected.
Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, transforming growth factor (TGF)-β, and IL-10 in the supernatants collected from brain tissue or microglia culture were detected by ELISA (Cusabio Co., Ltd., Wuhan, China). The standard working curves were generated according to the concentration range of each cytokine. Production of nitric oxide (NO) was determined by measuring the content of Nitrite (the end products of NO) in the media as previously described [15]. A standard Nitrite curve (0.25-50 μM) was generated using a 10 mM solution of NaNO2. All chemicals for the NO assay were from Sigma. Each sample was assayed in triplicate.

Quantitative real-time polymerase chain reaction
The total RNA of the sample was extracted by using a High-Capacity cDNA Archive Kit (Cat No.: 74804, QIA-GEN). Extracted RNAs with OD260/280 values from 1.8 to 2.1 were qualified for subsequent experiments. One microgram of the total RNA was reverse-transcribed into cDNA using a Rapid Reverse Transcription Kit (Cat No.: 11752-250, Invitrogen). The corresponding primers (Invitrogen, Table 1) and 50 ng cDNA were used to prepare PCR reaction solution with a fluorescent dye (RT2 SYBRr Green FAST Mastermixes, Cat No.: 330603, QIAGEN). qRT-PCR was performed in triplicate on a LightCycler 480 PCR machine (Applied Biosystemsr 7500 Real-Time PCR Systems, Thermo Fisher Scientific, Waltham, MA, USA) The thermal cycle parameters for qPCR were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 20 s, then 72°C for 20 s. The fluorescent signal was collected at 72°C for 20 s in the third step of each cycle. The cycle time was normalized to GAPDH in the same sample. The levels of mRNAs were normalized to GAPDH in the same sample and then reported as fold changes vs. Sham group.

Western blot
Proteins were isolated using a total protein extraction kit from Apply gen Technologies Inc., (Beijing, China). The protein concentration was determined using a BCA Protein Assay reagent kit (Novagen, Madison, WI, USA). Equal amounts of protein (100 μg) were separated by Tris-glycine SDS-PAGE and transferred to polyvinylidene fluoride (PVDF). The membranes were then incubated with blocking buffer (5% nonfat dry milk) at room temperature for 1 h and incubated with the followed primary antibodies overnight at 4°C: Rabbit polyclonal anti-GAPDH antibody (1:10000,Cell Signaling Technology, Danvers, MA, USA); Rabbit anti-β-catenin antibody (1:5000, Abcam); Mouse anti-GSK-3β antibody (1:1000, Abcam). Then, the membranes were incubated with the corresponding fluorescent labeling secondary antibody (1:8000, Rockland, Gilbertsville, PA, USA) for 1 h at room temperature. The relative density of each band was analyzed on an Odyssey infrared scanner (LICOR Bioscience, Lincoln, NE, USA). GAPDH was used as an internal control.

Tube formation assay
The bEnd.3 cells (2 × 10 4 per well) were seeded into 96well plates that were pre-coated with Matrigel (BD Biosciences Pharmingen, CA, USA). The 96-well plates were put in a chamber under normoxic condition for 12 h. After incubation, the numbers of the capillary-like structures were quantified by the ImageJ software in randomly selected five microscopic fields from each well.

CCK-8 assay
Cell Counting Kit 8 (CCK8, Dojindo, Japan) was used to evaluate cytotoxicity as previously described [30]. In brief, cell suspensions (1 × 10 4 cells, 100 μl/well) were seeded in a 96-well plate. Then, 10 μl of the CCK-8 reagent was added into each well, followed by 2 h of incubation at 37°C. The reaction was evaluated according to the absorbance at 450 nm.

Flow cytometry
Flow cytometry was used to detect the expression of CD16/32 and CD206 in microglia cells as previously described [31]. In brief, 100 μl microglia cell suspension (1 × 10 6 cells) was aliquoted into 1 ml Eppendorf tubes.

Statistical analysis
All outcome analysis was carried out by independent researchers blinded to the information of experimental groups. All data were presented as mean ± standard error of the mean (SEM) with the exception of the mNSS data, which were expressed as median ± interquartile range (IQR). Statistical analyses were performed using SPSS21.0 software (Version X, IBM, Armonk, NY, USA). Comparisons between two groups were evaluated using Student's t test (non-directional). Comparisons between multiple groups were analyzed with one-way or two-way analysis of variance (ANOVA) followed by Tukey's post hoc test, unless otherwise indicated. The data of mNSS were assessed using the Mann-Whitney rank-sum test. The Spearman or correlation was also determined. All analyses were two-tailed, and P < 0.05 was considered statistically significant.  Fig. 2f). As expected, compared with the vehicle group, mice in TWS119 group displayed lower mNSS at days 14 and 21 after stroke (Z = − 2.27, P = 0.023; Z = − 2.07, P = 0.038, Fig. 2 g). Collectively, the behavioral data suggested that TWS119 significantly promoted neurological recovery in the late phase of ischemic stroke. Additionally, TWS119 enhanced the level of β-catenin via inhibiting GSK-3β at day 14 after stroke and mitigated the histological damage of focal cerebral ischemic mouse at day 21 after stroke (see Supplementary Figure 2S, Additional file 1).

TWS119 facilitated post-stroke angiogenesis and neural plasticity
Neurovascular restoration is a multi-element exquisitely coordinated process, including angiogenesis and neural plasticity. The angiogenesis and neural plasticity were closely correlated with functional recovery following stroke [5]. To clarify the role of TWS119 on post-stroke angiogenesis, the expression of CD31 (cerebral microvascular endothelial cells marker) and BrdU (cell proliferation marker) were detected by immunofluorescence staining [32]. Compared with sham-operated mice, the vehicle mice displayed an elevation in area (3.91% ± 0.52% versus 7.05% ± 0.77%, P < 0.01; 5.88% ± 0.66% versus 8.90% ± 0.84%, P < 0.05, Fig. 3b) Fig. 3e). Collectively, the results indicated that TWS119 facilitated angiogenesis in the chronic phase of stroke.

Discussion
The limited neurorestorative capability contributes to long-term neurological deficits following ischemic stroke, which markedly impair the quality of a patient's daily life. The present study demonstrated that the Wnt canonical pathway activator TWS119 exhibits significant efficacy in the improvement of neurological function following experimental stroke, paralleled by an acceleration of angiogenesis and neural plasticity in the peri-infarct cortex. The underlying mechanism for the neurorestorative effect of TWS119 was probably due to β-catenin-mediated shift of microglia polarization from pro-inflammatory phenotype to anti-inflammatory phenotype.
An independent study [17] reported that intraperitoneal injection of TWS119 at 3.5 h decreased neurologic deficits, brain edema, infarct volume, and blood-brain barrier permeability at 24 h after stroke via activation the Wnt/β-catenin pathway in the ischemia-reperfusion rat model. To distinguish neuroprotective from neurorestorative effect on long-term neurological function, TWS119 was administered in a delayed manner in the current study. TWS119-treated mice displayed comprehensive improvement in long-term neurological function, including gait performance, sensorimotor function, postural asymmetry, motor coordination, and learning function. Angiogenesis and neural plasticity are key neurorestorative elements, and closely associated with poststroke behavioral and histological recovery [5]. Indeed, we observed that TWS119 improves neurological function and simultaneously facilitated angiogenesis and neural plasticity during the chronic phase of experimental stroke. Taken together, our findings indicated that the favorable effects of TWS119 on long-term poststroke outcomes could be attributed, at least partially, to the facilitated angiogenesis and neural plasticity.
TWS119 has a direct modulating effect on peripheral immune cell cells [18,19]. In present study, TWS119 modulated microglia/macrophages toward anti-inflammatory phenotype and attenuated inflammation in the peri-infarct cortex. In vitro, we found that the favorable effect of TWS119 on microglial polarization and microgliamediated inflammation status was accompanied with the enhancement of the level of β-catenin, and partly reversed by selective Wnt/β-catenin pathway blockade targeting Axin in the destruction complex of β-catenin. Additionally, our prior experiment confirmed TWS119 increased the expression of β-catenin in the peri-infarct cortex. Thus, we suggested that TWS119 modulated the switch of microglial polarization from pro-inflammatory phenotype to antiinflammatory phenotype following ischemic stroke probably via the Wnt/β-catenin pathway. In this mechanism, peroxisome proliferator-activated receptor-γ might be the downstream target of β-catenin, which has been shown in a hemorrhagic stroke model [16]. Furthermore, it is noteworthy that GSK-3β, the pharmacological target of TWS119, is a multifunctional kinase and is involved in multiple signaling pathways [36]. In the current study, we cannot exclude the involvement of other mechanisms in the effect of TWS119 on modulating microglial polarization after ischemic stroke. Nevertheless, our study offered a new mechanism on modulating microglial polarization, which enriched the understanding of the role of the Wnt/β-catenin signaling pathway in ischemia-induced neuroinflammation. We suggested that activation of the Wnt/β-catenin pathway during the late phase of ischemic stroke might be a worthwhile strategy on modulating microglial polarization to a protective phenotype. The M1/M2 terminology of microglia is widely debated [37]. Thus, the microglial phenotype was described as pro-inflammatory or antiinflammatory in the current study to avoid the controversy surrounding whether M1 and M2 polarized microglia exist.
Cerebral ischemia induces angiogenesis in the periinfarct cortex of ischemic mice, as does in ischemic stroke patients [38]. In the present study, TWS119 facilitated post-stroke angiogenesis in the ischemic brain, but failed to directly promote angiogenesis in OGD endothelial cells. Evidence revealed that microglia in anti-inflammatory phenotype promote angiogenesis [39]. Using a conditioned medium transfer system, we found that TWS119 promoted angiogenesis of OGD endothelial cells via anti-inflammatory phenotype microgliamediated amelioration of inflammatory microenvironment, suggesting a proangiogenic effect of TWS119 in anti-inflammatory phenotype polarization-dependent manner. In addition, there existed a spatial and temporal co-localization of angiogenesis, pro-inflammatory to anti-inflammatory switch of microglia phenotype, and amelioration of local inflammatory microenvironment in TWS119-treated mice. Altogether, we suggested that TWS119's efficacy on post-stroke angiogenesis was likely attributed to the local inflammatory microenvironment mediated by microglial polarization proinflammatory/ anti-inflammatory balance state. It is well known that vascular endothelial growth factor (VEGF) is the key pro-angiogenic factor for angiogenesis [40]. The Wnt/βcatenin pathway has been confirmed to promote poststroke angiogenesis via modulation on the expression of VEGF in a cerebral ischemia-reperfusion model [41]. Moreover, VEGF has been considered the upstream mechanism of microglial polarization [42]. In the current study, we could not rule out the possibility of VEGF involvement in the advantageous effect of TWS119 on post-stroke angiogenesis. Nevertheless, we indicated that TWS119 directly modulated microglia-mediated inflammatory status toward a better local microenvironment accelerated for neurovascular restoration following ischemic stroke. The neuroinflammation persists into the chronic phase of stroke and participates in all neurorestorative elements, including angiogenesis. Considering the significant correlation of anti-inflammatory cytokine IL-10 and neurological function in our study, we suggested modulation of neuroinflammatory responses shed promising light on neurological recovery following ischemic stroke.
Besides angiogenesis, neural plasticity, including the establishment of new synaptic contacts and sprouting of axonal projections, is another neurorestorative element following stroke. Microglia is involved in synaptic pruning by engulfing synaptic debris [43]. The M2 macrophage is a potent promoter of axonal sprouting by secreting the protective factor IL-10 [44]. In our study, TWS119 promoted neural plasticity accompanied by shift of microglial polarization from pro-inflammatory phenotype to anti-inflammatory phenotype in the periinfarct cortex at 21 days after ischemic stroke, suggesting an exquisite coordination of the two mechanisms. Thus, we speculated TWS119 stimulated neural plasticity might via modulating microglia toward antiinflammatory phenotype. However, contradictory evidence shows that microglia seem irrelevant to axonal regeneration following neuronal injury [45]. To clarify the role of microglia on neural plasticity following stroke, further investigation using primary neuron and primary microglia were needed in our future study.
The present research has several limitations that may impede the universalization of our findings in human ischemic stroke. The focal cerebral ischemic stroke animal model in our study mimic some of the key features of human ischemic stroke, including pathological progression and neurological deficits, but some risk factors of stroke, such as hypertension, diabetes, age, and gender, are not investigated in the present study. Additionally, we did not identify whether the Iba1 + cells originated from resident microglia or infiltration of blood macrophages. We only demonstrated that TWS119 modulated microglia/macrophages to anti-inflammatory phenotype in vivo. We will further identify the microglia and macrophages from the peri-infarct region of the brain using flow cytometry, in which microglia were stained as CD45 int CD11b +cells and macrophages stained as CD45 high CD11b + cells. Besides, the role of other immune cells involved in neuroinflammation, especially astrocytes, were not elucidated in our study. Whether Wnt/β-catenin pathway activator TWS119 has beneficial effect on astrocytes-mediated neuroinflammation need further investigation.

Conclusion
Wnt/β-catenin pathway activator TWS119 ameliorated neuroinflammatory microenvironment following experimental stroke via modulating pro-inflammatory to antiinflammatory phenotype switch of activated microglia and facilitates long-term neurological recovery in an antiinflammatory phenotype polarization-dependent manner. Activation of the Wnt/β-catenin pathway during the chronic phase of ischemic stroke might be a promising clinical strategy targeting neuroinflammation via modulating microglia toward anti-inflammatory phenotype and worth further evaluation.
Additional file 1. Supplementary result. TWS119 with the dose of 10 mg/kg was selected in animal experiment. TWS119 activated Wnt/βcatenin pathway by inhibiting GSK-3β. TWS119 improved histological damage in the late stage of stroke. Figure S1 TWS119 at the dose of 10 mg/kg was selected in animal experiment. a TWS119 with the dose of 5 mg/kg and TWS119 with the dose of 10 mg/kg upregulated the mRNA expression of β-catenin (n = 8 per group, * P < 0.05, ** P < 0.01). b Neurological functions were evaluated using Adhesive Removal test at day1, 7 and 14 after stroke. TWS119 with the dose of 10 mg/kg significantly improved neurological function at day 14 after stroke (n = 8 per group, TWS119 (10 mg/kg) vs Vehicle, # P < 0.01). Figure S2 TWS119 activated Wnt/β-catenin pathway by inhibiting GSK-3β and improved histological damage. a Western blot was used to determine the expression of β-catenin and GSK-3β 14 days after stroke. b and c TWS119-treated mice had a lower level of GSK-3β and a higher level β-catenin comparing to vehicle mice (n = 6 per group, * P < 0.05, ** P < 0.01). d Histological damage was assessed by quantification of the infarcted cortex cavitation using CWI. e TWS119 treatment increased the CWI in ischemic mice comparing to vehicle treatment 21 days after stroke (n = 8 per group, * P < 0.05). CWI, Cortical width index. CWI = 100% × W.lpsi / W.contra, W.lpsi means ipsilateral width, W.contra means contralateral width.