Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke methods
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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
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Gait recovery and locomotor function
- 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
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- Group comparisons of gait indices, stride metrics, or recovery curves
Protein expression by western blot
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- Membrane or gel image with visible bands for target and control proteins
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- Band quantification and normalized densitometry values
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- Relative expression values or fold-change comparisons across groups
Analysis plan
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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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