Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke methods
Aim. Evidence-backed execution summary for Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke methods from Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- 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 [ ]. Intraperitoneal injection followed the aseptic principle and recommended procedures to avoid complicatio...
Primary microglia
reagent used in the protocol.
- Use
- Primary microglia cells were prepared from the cerebral cortex of neonatal C57BL/6 mice at P1-P2 as previously reported with some modifications [ ]. 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% peni...
Primary microglia
reagent used in the protocol.
- Use
- For pro-inflammatory phenotype polarization, serum-starved 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 [ ].
Primary microglia
reagent used in the protocol.
- Use
- 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...
Mouse brain microvascular endothelial cell line
reagent used in the protocol.
- Use
- The mouse brain microvascular endothelial cell line bEnd.3 (American Type Culture Collection (ATCC), Manassas, VA, USA) were used to evaluate angiogenesis in vitro.
Mouse brain microvascular endothelial cell line
reagent used in the protocol.
- Use
- Pro-angiogenic structures of OGD bEnd.3 cells in DMEM and CM (microglia supernatant) were detected by tube formation assay [ ]. Firstly, the bEnd.3 cells were divided into 3 groups and cultured in DMEM: bEnd.3: bEnd.3 cells without OGD injure and TWS119 treatment; OGDbEnd.3: bEnd.3 cells were subjected to OGD injure...
Immunofluorescence staining
reagent used in the protocol.
- Use
- Mice were deeply anesthetized and then fixed by transcardial perfusion with cold saline followed by 4% paraformaldehyde. Coronal slices (15-µm thick) were obtained from the brain blocks centered on the ischemic lesion (approximately bregma - 1.5 mm to + 0.5 mm) using a cryostat microtome (LEIC...
Enzyme-linked immunosorbent assay and Griess reaction
reagent used in the protocol.
- Use
- 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 peri-infract region; for slices on day 21,...
Methods
Ischemic stroke was induced in male mice (10 weeks old) by permanent distal middle cerebral artery (MCA) occlusion plus 1 h hypoxia [ ]. 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 an...
- Use
- Ischemic stroke was induced in male mice (10 weeks old) by permanent distal middle cerebral artery (MCA) occlusion plus 1 h hypoxia [ ]. 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 an...
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.
- Use
- 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.
Neurobehavioral test
CatWalkXT (v10.6, Noldus Information Technology) test was used to measure Walking performance of ischemic mice as described previously [ ]. 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 defi...
- Use
- CatWalkXT (v10.6, Noldus Information Technology) test was used to measure Walking performance of ischemic mice as described previously [ ]. 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 defi...
Neurobehavioral test
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 [ ]. The mice were placed on a gradually accelerated rotating rod (4 to 10 rpm, 5 min). Re...
- Use
- 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 [ ]. The mice were placed on a gradually accelerated rotating rod (4 to 10 rpm, 5 min). Re...
Histological assessment
Cortical width index (CWI) was used to assess histological damage as described previously [ ]. 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....
- Use
- Cortical width index (CWI) was used to assess histological damage as described previously [ ]. 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....
Immunofluorescence staining
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 [ ]. CD31-positive blood vessels were highlighted using the...
- Use
- 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 [ ]. CD31-positive blood vessels were highlighted using the...
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, QIAGEN). 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 N...
- Use
- The total RNA of the sample was extracted by using a High-Capacity cDNA Archive Kit (Cat No.: 74804, QIAGEN). 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 N...
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 transfer...
- Use
- 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 transfer...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- 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 pe...
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Methods
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.
Methods
Ischemic stroke was induced in male mice (10 weeks old) by permanent distal middle cerebral artery (MCA) occlusion plus 1 h hypoxia [ ]. 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 low-heat 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 he...
Methods
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 [ ]. 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. a. Brain tissue for protein, gene, and cytokine detection were harvested from the peri-i...
Neurobehavioral test
CatWalkXT (v10.6, Noldus Information Technology) test was used to measure Walking performance of ischemic mice as described previously [ ]. 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.
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...
TWS119 promoted pro-inflammatory to anti-inflammatory phenotype switch of microglia probably via the Wnt/β-caten...
Primary microglia were used to further determine the modulating effect of TWS119 on microglial polarization and elucidate the underlying role of Wnt/β-catenin pathway in this. Pro-inflammatory polarized microglia were induced by LPS plus IFN-γ stimulation in vitro. Firstly, we selected the effective dose of TWS119 in microglia. CCK-8 assay was performed to determine the cytotoxicity of TWS119; TWS119 with doses (≥ 100 µM) exerted cytotoxic effect on microglia cells (Fig. a). TWS119 with doses (10 µM, 20 µM) increased the secretion of IL-10 in microglia cells stimulated with LPS+IFN-γ ( P = 0.002; P = 0.001, Fig. b), while TWS119 with doses (5 µM, 40 µM) had no similar effect. TWS119 at the dose of 10 µM is widely used in other cell lines [, ], Thus, the dose (10 µM) was used in our experiment...
Supplementary information
Additional file 1. Supplementary result. TWS119 with the dose of 10 mg/kg was selected in animal experiment. TWS119 activated Wnt/β-catenin 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....
Measurement outputs
What raw and processed outputs should exist?
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...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
CatWalkXT (v10.6, Noldus Information Technology) test was used to measure Walking performance of ischemic mice as described previously [ ]. Each mouse walked freely through a co...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Accelerated Rotarod (Ugo Basile 47600, Ugo-Basile, Biological Research Apparatus, Milan, Italy) test was performed to evaluate motor coordination and learning function of ischem...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Modified neurological severity scores (mNSS) were performed to evaluate functional impairments [ ]. The scores were graded on a scale ranging from 0 to 18 (normal score 0, maxim...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
All outcome analysis was carried out by independent researchers blinded to the information of experimental groups.
from paperScoring or quantification
Quantify the primary readouts for this experiment: 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...; CatWalkXT (v10.6, Noldus Information Technology) test was used to measure Walking performance of ischemic mice as described previously [ ]. Each mouse walked freely through a co...; Accelerated Rotarod (Ugo Basile 47600, Ugo-Basile, Biological Research Apparatus, Milan, Italy) test was performed to evaluate motor coordination and learning function of ischem...; Modified neurological severity scores (mNSS) were performed to evaluate functional impairments [ ]. The scores were graded on a scale ranging from 0 to 18 (normal score 0, maxim....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
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 m...; Neurovascular restoration is a multi-element exquisitely coordinated process, including angiogenesis and neural plasticity. The angiogenesis and neural plasticity were closely c...; Primary microglia were used to further determine the modulating effect of TWS119 on microglial polarization and elucidate the underlying role of Wnt/β-catenin pathway in th...; The bEnd.3 cell line was used to explore the underlying mechanism on the effect of TWS119 on promoting post-stroke angiogenesis. OGD injury was mimicked cerebral ischemic injury...
from paperReporting output
Report representative outputs alongside summary comparisons for 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..., CatWalkXT (v10.6, Noldus Information Technology) test was used to measure Walking performance of ischemic mice as described previously [ ]. Each mouse walked freely through a co..., Accelerated Rotarod (Ugo Basile 47600, Ugo-Basile, Biological Research Apparatus, Milan, Italy) test was performed to evaluate motor coordination and learning function of ischem..., Modified neurological severity scores (mNSS) were performed to evaluate functional impairments [ ]. The scores were graded on a scale ranging from 0 to 18 (normal score 0, maxim....
inferred from protocolStructured statistical methods
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 m...; Neurovascular restoration is a multi-element exquisitely coordinated process, including angiogenesis and neural plasticity. The angiogenesis and neural plasticity were closely c...; Primary microglia were used to further determine the modulating effect of TWS119 on microglial polarization and elucidate the underlying role of Wnt/β-catenin pathway in th...; The bEnd.3 cell line was used to explore the underlying mechanism on the effect of TWS119 on promoting post-stroke angiogenesis. OGD injury was mimicked cerebral ischemic injury...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (7)
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 [ ]. 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 low-heat 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. Sham-operated mice...
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 [ ]. 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. a. Brain tissue for protein, gene, and cytokine detection were harvested from the peri-infarct region as described previously [ ]. 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. b. Fig. 1 Experimental o...
CatWalkXT (v10.6, Noldus Information Technology) test was used to measure Walking performance of ischemic mice as described previously [ ]. 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.
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.
Primary microglia were used to further determine the modulating effect of TWS119 on microglial polarization and elucidate the underlying role of Wnt/β-catenin pathway in this. Pro-inflammatory polarized microglia were induced by LPS plus IFN-γ stimulation in vitro. Firstly, we selected the effective dose of TWS119 in microglia. CCK-8 assay was performed to determine the cytotoxicity of TWS119; TWS119 with doses (≥ 100 µM) exerted cytotoxic effect on microglia cells (Fig. a). TWS119 with doses (10 µM, 20 µM) increased the secretion of IL-10 in microglia cells stimulated with LPS+IFN-γ ( P = 0.002; P = 0.001, Fig. b), while TWS119 with doses (5 µM, 40 µM) had no similar effect. TWS119 at the dose of 10 µM is widely used in other cell lines [, ], Thus, the dose (10 µM) was used in our experiment in vitro. The dose (10 µM) of IWR-1 used in microglia cells was according to previous research [, ]. Next, qRT-PCR was used to characterize the polarization of microglia cells (Fig. c). The mRNA level of CD16 was drastically upregulated in microglia cells stimulated by LPS plus IFN-γ...
Additional file 1. Supplementary result. TWS119 with the dose of 10 mg/kg was selected in animal experiment. TWS119 activated Wnt/β-catenin 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 damag...
Machine-readable layer
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"name": "Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke methods",
"description": "Evidence-backed execution summary for Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke methods from Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke.",
"step": [
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"text": "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."
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"text": "Ischemic stroke was induced in male mice (10 weeks old) by permanent distal middle cerebral artery (MCA) occlusion plus 1 h hypoxia [ ]. 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 low-heat 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 he..."
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"text": "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 [ ]. 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. a. Brain tissue for protein, gene, and cytokine detection were harvested from the peri-i..."
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"text": "CatWalkXT (v10.6, Noldus Information Technology) test was used to measure Walking performance of ischemic mice as described previously [ ]. 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."
},
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"name": "Western blot",
"text": "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..."
},
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"name": "TWS119 promoted pro-inflammatory to anti-inflammatory phenotype switch of microglia probably via the Wnt/β-caten...",
"text": "Primary microglia were used to further determine the modulating effect of TWS119 on microglial polarization and elucidate the underlying role of Wnt/β-catenin pathway in this. Pro-inflammatory polarized microglia were induced by LPS plus IFN-γ stimulation in vitro. Firstly, we selected the effective dose of TWS119 in microglia. CCK-8 assay was performed to determine the cytotoxicity of TWS119; TWS119 with doses (≥ 100 µM) exerted cytotoxic effect on microglia cells (Fig. a). TWS119 with doses (10 µM, 20 µM) increased the secretion of IL-10 in microglia cells stimulated with LPS+IFN-γ ( P = 0.002; P = 0.001, Fig. b), while TWS119 with doses (5 µM, 40 µM) had no similar effect. TWS119 at the dose of 10 µM is widely used in other cell lines [, ], Thus, the dose (10 µM) was used in our experiment..."
},
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"@type": "HowToStep",
"position": 7,
"name": "Supplementary information",
"text": "Additional file 1. Supplementary result. TWS119 with the dose of 10 mg/kg was selected in animal experiment. TWS119 activated Wnt/β-catenin 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...."
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