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
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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?
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
Analysis plan
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Acquisition
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Quantify the primary readouts for this experiment: Gait recovery and locomotor function.
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inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function.
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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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