Intermittent theta-burst stimulation improves motor function by inhibiting neuronal pyroptosis and regulating microglial polarization via TLR4/NFκB/NLRP3 signaling pathway in cerebral ischemic mice methods
Aim. Evidence-backed execution summary for Intermittent theta-burst stimulation improves motor function by inhibiting neuronal pyroptosis and regulating microglial polarization via TLR4/NFκB/NLRP3 signaling pathway in cerebral ischemic mice methods from Intermittent theta-burst stimulation improves motor function by inhibiting neuronal pyroptosis and regulating microglial polarization via TLR4/NFκB/NLRP3 signaling pathway in cerebral ischemic mice.
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mouse
Subject model for the experiment.
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Methods
reagent used in the protocol.
- Use
- We evaluated the motor function in middle cerebral artery occlusion/reperfusion (MCAO/r) injury mice after 1-week intermittent theta-burst rTMS (iTBS) treatment in the early phase with or without depletion of microglia by colony-stimulating factor 1 receptor (CSF1R) inhibitor treatment, respectively. We further expl...
Colony-stimulating factor 1 receptor (CSF1R) inhibitor treatment
reagent used in the protocol.
- Use
- To deplete microglia in vivo, mice received the CSF1R inhibitor PLX3397 (AdooQ BioScience, A15520), which were mixed into AIN-76A standard chow at 290 mg/kg. AIN-76A standard chow alone served as the control. Mice were placed on the chow diet ad libitum for 3 weeks prior to induction of MCAO/r and the diet...
Experimental material preparation
reagent used in the protocol.
- Use
- Mice were deeply anesthetized by 1% pentobarbital sodium (100 mg/kg. ip). For western blotting, cytokine bioassays, RNA sequencing, mice in each group were sacrificed and brain tissue of the infarcted cortex were quickly removed, placed in Eppendorf tubes, frozen in liquid nitrogen, and stored at - 8...
Western blot assays
reagent used in the protocol.
- Use
- Brain tissue was homogenized with an electric homogenizer in RIPA lysis buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0). Centrifuge for 10 min at 12,000 rpm at 4 °C in a microcentrifuge and aspirate the supernatant. Protein conc...
Nissl staining
reagent used in the protocol.
- Use
- Nissl staining was performed as previously described [ ]. Briefly, sections were degreased through graded alcohol (70%, 95% and 100% alcohol) for 3 min, respectively, and then hydrated through graded alcohol (95%, 70% and 50% alcohol) for 3 min, respectively. Subsequently, the sections were stained in 0.1%...
Immunofluorescence staining
reagent used in the protocol.
- Use
- For immunofluorescence staining, the peri-infarcted brain area of coronal slice was chosen for imaging and analysis. Briefly, coronal cryotome section (30 µm) were washed with 0.5% Triton X-100 for 10 min, and blocked with 5% bovine serum albumin (BSA) (Sigma) and 0.5% Triton X-100 (Sigma) in PBS...
RNA sequencing and differentially expressed genes analysis
reagent used in the protocol.
- Use
- Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA purity and quantification were evaluated using the NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies...
5-Ethynyl-2′-deoxyuridine (EdU) labeling, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling...
reagent used in the protocol.
- Use
- To examine cell proliferation, EdU (50 mg/kg body weight, Beyotime, China) was intraperitoneally administered to each animal every 2 days over the 8-day period, initiated 24 h after MCAO/r surgery. EdU staining was performed with the Edu-488 cell proliferation detection kit (Beyotime, China). Briefly,...
Methods
We evaluated the motor function in middle cerebral artery occlusion/reperfusion (MCAO/r) injury mice after 1-week intermittent theta-burst rTMS (iTBS) treatment in the early phase with or without depletion of microglia by colony-stimulating factor 1 receptor (CSF1R) inhibitor treatment, respectively. We further expl...
- Use
- We evaluated the motor function in middle cerebral artery occlusion/reperfusion (MCAO/r) injury mice after 1-week intermittent theta-burst rTMS (iTBS) treatment in the early phase with or without depletion of microglia by colony-stimulating factor 1 receptor (CSF1R) inhibitor treatment, respectively. We further expl...
Behavioral tests
The behavioral tests were performed 1 day before and 8 days after MCAO/r surgery. The test sequence was as follows: (1) Open field test; (2) Rotarod test; (3) Cylinder test; (4) Inverted wire mesh grid grip test; (5) CatWalk XT gait test. Keeping a 2-h interval between each test.
- Use
- The behavioral tests were performed 1 day before and 8 days after MCAO/r surgery. The test sequence was as follows: (1) Open field test; (2) Rotarod test; (3) Cylinder test; (4) Inverted wire mesh grid grip test; (5) CatWalk XT gait test. Keeping a 2-h interval between each test.
Behavioral tests
Open field test The experimental device consists of a plastic test box (40 × 40 × 50 cm) and an automatic video system. The mouse was placed into the center of the open field to record its free movement for 5 min. The distance moved and mean velocity were recorded and anal...
- Use
- Open field test The experimental device consists of a plastic test box (40 × 40 × 50 cm) and an automatic video system. The mouse was placed into the center of the open field to record its free movement for 5 min. The distance moved and mean velocity were recorded and anal...
Middle cerebral artery occlusion/reperfusion (MCAO/r) surgery
MCAO/r model of left middle cerebral artery ischemia for 60 min was produced as previously described [ ]. Briefly, male C57BL/6 J mice were anesthetized with 1% pentobarbital sodium (100 mg/kg. ip). The body temperature was maintained at 37.0 ± 0.3 °C during surgery via a te...
- Use
- MCAO/r model of left middle cerebral artery ischemia for 60 min was produced as previously described [ ]. Briefly, male C57BL/6 J mice were anesthetized with 1% pentobarbital sodium (100 mg/kg. ip). The body temperature was maintained at 37.0 ± 0.3 °C during surgery via a te...
Repetitive transcranial magnetic stimulation application
Mice were randomly assigned to three groups: (1) Sham group ( n = 15); (2) MCAO/r group ( n = 25); (3) iTBS group ( n = 25). During the stimulation, mice were un-anesthetized, gently grasped in rubber glove (Fig. C). To reduce stress related to handling, restraining, and...
- Use
- Mice were randomly assigned to three groups: (1) Sham group ( n = 15); (2) MCAO/r group ( n = 25); (3) iTBS group ( n = 25). During the stimulation, mice were un-anesthetized, gently grasped in rubber glove (Fig. C). To reduce stress related to handling, restraining, and...
Behavioral tests
Rotarod test The pre-training test (to establish a performance baseline) was performed 4 times per day for 3 consecutive days. The rotating rod accelerates from 5 revolutions per minute (RPM) until it rotates at a constant speed after 40 RPM within 90 s. Each test lasts for a maximum of 5 min, and rests fo...
- Use
- Rotarod test The pre-training test (to establish a performance baseline) was performed 4 times per day for 3 consecutive days. The rotating rod accelerates from 5 revolutions per minute (RPM) until it rotates at a constant speed after 40 RPM within 90 s. Each test lasts for a maximum of 5 min, and rests fo...
Behavioral tests
Cylinder test Mouse was placed in a transparent cylinder (10 cm in diameter and 15 cm in height), and record the use of forelimbs in the exploratory behavior within 5 min. Place a mirror at the appropriate position to ensure that the forelimb activity can be recorded even when the mouse was turned bac...
- Use
- Cylinder test Mouse was placed in a transparent cylinder (10 cm in diameter and 15 cm in height), and record the use of forelimbs in the exploratory behavior within 5 min. Place a mirror at the appropriate position to ensure that the forelimb activity can be recorded even when the mouse was turned bac...
Behavioral tests
CatWalk XT gait test In this study, a 150 cm-long small animal glass track CatWalk XT (Noldus, Netherlands, Wageningen) system was used. A high-speed camera was placed under the glass plate to capture images of mouse paw changes for gait data collection. In the experiment, the mouse was placed on the runway and...
- Use
- CatWalk XT gait test In this study, a 150 cm-long small animal glass track CatWalk XT (Noldus, Netherlands, Wageningen) system was used. A high-speed camera was placed under the glass plate to capture images of mouse paw changes for gait data collection. In the experiment, the mouse was placed on the runway and...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical tests were done on SPSS 23.0 statistical software (SPSS, Chicago, IL, USA) and GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA) using one-way or two-way analysis of variance (ANOVA) for multiple comparisons followed by Tukey's or Bonferroni's post hoc test. Statistical signific...
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Behavioral tests
The behavioral tests were performed 1 day before and 8 days after MCAO/r surgery. The test sequence was as follows: (1) Open field test; (2) Rotarod test; (3) Cylinder test; (4) Inverted wire mesh grid grip test; (5) CatWalk XT gait test. Keeping a 2-h interval between each test.
Materials and methods
Adult male C57BL/6 J mice (20-25 g) were purchased from Shanghai Jihui Laboratory Animal Care Co., Ltd., and housed in constant room temperature (22 ± 2 °C) with 50 ± 10% humidity under a 12/12 h light/dark cycle (lights on 7:00 a.m.) with food and water ad libitum for at least 1 week before the experiments. All experiments procedures were conducted according to the Animal Care and Use Committee guidelines of Huashan Hospital of Fudan University. Every effort was made to avoid/alleviate animal pain and distress and minimize the number of animals used in the project.
Behavioral tests
Open field test The experimental device consists of a plastic test box (40 × 40 × 50 cm) and an automatic video system. The mouse was placed into the center of the open field to record its free movement for 5 min. The distance moved and mean velocity were recorded and analyzed [ ].
Middle cerebral artery occlusion/reperfusion (MCAO/r) surgery
MCAO/r model of left middle cerebral artery ischemia for 60 min was produced as previously described [ ]. Briefly, male C57BL/6 J mice were anesthetized with 1% pentobarbital sodium (100 mg/kg. ip). The body temperature was maintained at 37.0 ± 0.3 °C during surgery via a temperature-regulated heating pad. Incisions were made at the neck midline to expose the left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). After ECA was ligated, a silicon-coated monofilament (diameter: 0.18 ± 0.01 mm, Guangzhou Jialing Co., Ltd, China) was inserted from the left ECA, through the bifurcation of CCA, into the intracranial segment of ICA (10 ± 1 mm away from the carotid bifurcation) to block blood flow at middle cerebral artery (MCA) for 60 min. The regional cerebr...
Neurobehavioral assessment
The neurological deficit level of the mice was examined using the modified neurological severity score (mNSS) 24 h after MCAO/r surgery. The full score of mNSS is 14 points, including motor (muscle state and abnormal movement), reflex and balance tests. Failure to perform one of the tests will get 1 point, and no corresponding test reflection will deduct 1 point. The overall comprehensive score is used to determine the degree of injury. MCAO/r injured mice with mNSS more than 6 points participated in the experiment.
Behavioral tests
Rotarod test The pre-training test (to establish a performance baseline) was performed 4 times per day for 3 consecutive days. The rotating rod accelerates from 5 revolutions per minute (RPM) until it rotates at a constant speed after 40 RPM within 90 s. Each test lasts for a maximum of 5 min, and rests for 3 min between tests to avoid fatigue. Record the latency of each mouse falling from the rod in each test and calculate the average latency as the final result. If the mouse grabs the revolver for 2 laps, stop timing [ ].
Behavioral tests
Cylinder test Mouse was placed in a transparent cylinder (10 cm in diameter and 15 cm in height), and record the use of forelimbs in the exploratory behavior within 5 min. Place a mirror at the appropriate position to ensure that the forelimb activity can be recorded even when the mouse was turned back to the camera lens. The evaluator used a video recorder with slow-motion and clear freeze-frame functions to record and score. When analyzing the behavior in the cylinder, record the number of times that the right paw, the left paw, and both paws touch the cylinder wall simultaneously. Data were analyzed using asymmetry, which was calculated as follows: (left + 0.5 × both)/(right + left + both) × 100% [ ].
Behavioral tests
Inverted wire mesh grid grip test Mouse was gently placed on a metal wire mesh grid with 0.8 cm gaps (12 mm in square, 1 mm in diameter). After the mice stood steadily, the mesh was rotated slowly at a steady speed to an inverted position and suspended 40 cm above a cage. Record the latency of each mouse falling from the mesh. Each testing session included 3 trials with a 5-min delay interval, and calculate the average latency as the final result [ ].
Measurement outputs
What raw and processed outputs should exist?
We evaluated the motor function in middle cerebral artery occlusion/reperfusion (MCAO/r) injury mice after 1-week intermittent theta-burst rTMS (iTBS) treatment in the early pha...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Open field test The experimental device consists of a plastic test box (40 × 40 × 50 cm) and an automatic video system. The mouse was plac...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
The neurological deficit level of the mice was examined using the modified neurological severity score (mNSS) 24 h after MCAO/r surgery. The full score of mNSS is 14 points...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Rotarod test The pre-training test (to establish a performance baseline) was performed 4 times per day for 3 consecutive days. The rotating rod accelerates from 5 revolutions pe...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Statistical tests were done on SPSS 23.0 statistical software (SPSS, Chicago, IL, USA) and GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA) using one-way or two-way analysis of variance (ANOVA) for multiple comparisons followed by Tukey's or BonferroniR...
from paperScoring or quantification
Quantify the primary readouts for this experiment: We evaluated the motor function in middle cerebral artery occlusion/reperfusion (MCAO/r) injury mice after 1-week intermittent theta-burst rTMS (iTBS) treatment in the early pha...; Open field test The experimental device consists of a plastic test box (40 × 40 × 50 cm) and an automatic video system. The mouse was plac...; The neurological deficit level of the mice was examined using the modified neurological severity score (mNSS) 24 h after MCAO/r surgery. The full score of mNSS is 14 points...; Rotarod test The pre-training test (to establish a performance baseline) was performed 4 times per day for 3 consecutive days. The rotating rod accelerates from 5 revolutions pe....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Statistical tests were done on SPSS 23.0 statistical software (SPSS, Chicago, IL, USA) and GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA) using one-way or two-w...; Nissl staining showed that neuronal cells in the MCAO/r group were loosely arranged or missing, and Nissl bodies were lightly stained or even dissolved compared with that in Sha...; Different from apoptosis and necrosis, pyroptosis is emerging as a common innate immune effector mechanism in vertebrates [ ]. Innate immunity requires the pattern recognition r...; We profiled the levels of 13 cytokines, using the bead-based immunoassay LEGENDplex, to explore the local inflammatory status of infarcted tissue. Notably, 5 out of 13 cytokines...
from paperReporting output
Report representative outputs alongside summary comparisons for We evaluated the motor function in middle cerebral artery occlusion/reperfusion (MCAO/r) injury mice after 1-week intermittent theta-burst rTMS (iTBS) treatment in the early pha..., Open field test The experimental device consists of a plastic test box (40 × 40 × 50 cm) and an automatic video system. The mouse was plac..., The neurological deficit level of the mice was examined using the modified neurological severity score (mNSS) 24 h after MCAO/r surgery. The full score of mNSS is 14 points..., Rotarod test The pre-training test (to establish a performance baseline) was performed 4 times per day for 3 consecutive days. The rotating rod accelerates from 5 revolutions pe....
inferred from protocolStructured statistical methods
Statistical tests were done on SPSS 23.0 statistical software (SPSS, Chicago, IL, USA) and GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA) using one-way or two-w...; Nissl staining showed that neuronal cells in the MCAO/r group were loosely arranged or missing, and Nissl bodies were lightly stained or even dissolved compared with that in Sha...; Different from apoptosis and necrosis, pyroptosis is emerging as a common innate immune effector mechanism in vertebrates [ ]. Innate immunity requires the pattern recognition r...; We profiled the levels of 13 cytokines, using the bead-based immunoassay LEGENDplex, to explore the local inflammatory status of infarcted tissue. Notably, 5 out of 13 cytokines...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
The behavioral tests were performed 1 day before and 8 days after MCAO/r surgery. The test sequence was as follows: (1) Open field test; (2) Rotarod test; (3) Cylinder test; (4) Inverted wire mesh grid grip test; (5) CatWalk XT gait test. Keeping a 2-h interval between each test.
Adult male C57BL/6 J mice (20-25 g) were purchased from Shanghai Jihui Laboratory Animal Care Co., Ltd., and housed in constant room temperature (22 ± 2 °C) with 50 ± 10% humidity under a 12/12 h light/dark cycle (lights on 7:00 a.m.) with food and water ad libitum for at least 1 week before the experiments. All experiments procedures were conducted according to the Animal Care and Use Committee guidelines of Huashan Hospital of Fudan University. Every effort was made to avoid/alleviate animal pain and distress and minimize the number of animals used in the project.
Open field test The experimental device consists of a plastic test box (40 × 40 × 50 cm) and an automatic video system. The mouse was placed into the center of the open field to record its free movement for 5 min. The distance moved and mean velocity were recorded and analyzed [ ].
MCAO/r model of left middle cerebral artery ischemia for 60 min was produced as previously described [ ]. Briefly, male C57BL/6 J mice were anesthetized with 1% pentobarbital sodium (100 mg/kg. ip). The body temperature was maintained at 37.0 ± 0.3 °C during surgery via a temperature-regulated heating pad. Incisions were made at the neck midline to expose the left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). After ECA was ligated, a silicon-coated monofilament (diameter: 0.18 ± 0.01 mm, Guangzhou Jialing Co., Ltd, China) was inserted from the left ECA, through the bifurcation of CCA, into the intracranial segment of ICA (10 ± 1 mm away from the carotid bifurcation) to block blood flow at middle cerebral artery (MCA) for 60 min. The regional cerebral blood flow (rCBF) of MCA was monitored by laser speckle flowmetry (RWD life science, Shenzhen, China; Additional file: Fig. S1B). A decline in ischemic rCBF ≥ 70% of the contralateral side during surgery was considered as a successful occlusion (Fig. A, B). Then, the mono...
The neurological deficit level of the mice was examined using the modified neurological severity score (mNSS) 24 h after MCAO/r surgery. The full score of mNSS is 14 points, including motor (muscle state and abnormal movement), reflex and balance tests. Failure to perform one of the tests will get 1 point, and no corresponding test reflection will deduct 1 point. The overall comprehensive score is used to determine the degree of injury. MCAO/r injured mice with mNSS more than 6 points participated in the experiment.
Rotarod test The pre-training test (to establish a performance baseline) was performed 4 times per day for 3 consecutive days. The rotating rod accelerates from 5 revolutions per minute (RPM) until it rotates at a constant speed after 40 RPM within 90 s. Each test lasts for a maximum of 5 min, and rests for 3 min between tests to avoid fatigue. Record the latency of each mouse falling from the rod in each test and calculate the average latency as the final result. If the mouse grabs the revolver for 2 laps, stop timing [ ].
Cylinder test Mouse was placed in a transparent cylinder (10 cm in diameter and 15 cm in height), and record the use of forelimbs in the exploratory behavior within 5 min. Place a mirror at the appropriate position to ensure that the forelimb activity can be recorded even when the mouse was turned back to the camera lens. The evaluator used a video recorder with slow-motion and clear freeze-frame functions to record and score. When analyzing the behavior in the cylinder, record the number of times that the right paw, the left paw, and both paws touch the cylinder wall simultaneously. Data were analyzed using asymmetry, which was calculated as follows: (left + 0.5 × both)/(right + left + both) × 100% [ ].
Inverted wire mesh grid grip test Mouse was gently placed on a metal wire mesh grid with 0.8 cm gaps (12 mm in square, 1 mm in diameter). After the mice stood steadily, the mesh was rotated slowly at a steady speed to an inverted position and suspended 40 cm above a cage. Record the latency of each mouse falling from the mesh. Each testing session included 3 trials with a 5-min delay interval, and calculate the average latency as the final result [ ].
Machine-readable layer
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"datePublished": "2022",
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