Source Paper
Inhibition of Acyl-CoA Synthetase Long-Chain Family Member 4 Facilitates Neurological Recovery After Stroke by Regulation Ferroptosis
Junmin Chen, Lan Yang, et al.
Source Paper
Junmin Chen, Lan Yang, et al.
Frontiers in Cellular Neuroscience • 2021
Background Ischemic stroke is the main cause of disability worldwide, leading to a serious socioeconomic burden. Ferroptosis is a non-apoptotic form of programmed cell death and is related to various diseases. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is considered a target of ferroptosis, but its specific role in ischemic stroke remains unclear. In this study, we investigate whether the inhibition of ACSL4 promotes the recovery of neurological function in a way that prevents ferroptosis. Methods A transient cerebral ischemia model was established for mice by middle cerebral artery occlusion (MCAO); glutathione peroxidase 4 (GPx4), ACSL4 and cyclooxygenase 2 (COX2) were detected by Western blot, and changes to mitochondria were observed by a transmission electron microscope. A kit was used to determine iron levels and lipid peroxide indicators, such as glutathione peroxidase (GPx), reduced glutathione (GSH), total glutathione/oxidized glutathione (GSH/GSSG), lipid peroxidation, reactive oxygen species, superoxide and malonaldehyde. Following MCAO, a ferroptosis inhibitor, liproxstatin-1, was administered intranasally immediately at a concentration of 10 mg/kg. Rosiglitazone was used to inhibit ACSL4 and was administered intravenously 1 h before MCAO at a concentration of 0.4 mg/kg. Brain injury was determined by neurological deficit scores, neuroscore (28-point), corner test and gait analyses, at 24 and 72 h after stroke. Brain infarct volume was determined by 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining at 72 h after stroke. Results After MCAO, GPx4 protein expression decreased, ACSL4 and COX2 protein expression increased, GPx activity decreased and iron accumulation. Transmission electron microscopy confirmed that the outer mitochondrial membrane of neurons had ruptured and mitochondrial cristae had decreased or disappeared. Liproxstatin-1 could significantly attenuate the decrease of GPx4 and the increase of COX2 after MCAO, dramatically reducing iron accumulation and decreasing GPx activity, accompanied by a marked reduction in changes in lipid peroxidation indicators. The use of rosiglitazone to inhibit ACSL4 could significantly improve neurological function and reduce the brain infarct volume at 72 h after stroke. Importantly, inhibiting ACSL4 could significantly attenuate the decline of GPx4 after MCAO and markedly attenuate iron accumulation and a decrease in GPx activity. Additionally, changes in lipid peroxidation indicators were also significantly inhibited. Conclusion This study indicates that inhibiting ACSL4 can promote the recovery of neurological function after stroke by suppression of ferroptosis.
Objective: Assessment of brain injury using neurological deficit scores, neuroscore, corner test, and gait analysis following transient cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in mice
This is a Neurological Deficit Scoring protocol using mouse as the model organism. The procedure involves 12 procedural steps, 1 equipment items, 3 materials. Extracted from a 2021 paper published in Frontiers in Cellular Neuroscience.
Model and subjects
mouse • Not specified • unknown • Not specified • Not specified
Study window
~433 hours hands-on
Core workflow
Establish transient cerebral ischemia model • Administer Rosiglitazone • Administer Liproxstatin-1
Primary readouts
Key equipment and reagents
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Perform middle cerebral artery occlusion (MCAO) in mice to induce transient cerebral ischemia
Note: This is the baseline injury model
“A transient cerebral ischemia model was established for mice by middle cerebral artery occlusion (MCAO)”
Administer Rosiglitazone intravenously to inhibit ACSL4 prior to MCAO
Note: ACSL4 inhibitor given as preventive treatment
“Rosiglitazone was used to inhibit ACSL4 and was administered intravenously 1 h before MCAO at a concentration of 0.4 mg/kg”
Administer ferroptosis inhibitor liproxstatin-1 intranasally immediately following MCAO
Note: Intranasal administration route used
“Following MCAO, a ferroptosis inhibitor, liproxstatin-1, was administered intranasally immediately at a concentration of 10 mg/kg”
Assess brain injury using neurological deficit scores at 24 and 72 hours post-stroke
Note: Specific scoring methodology not detailed in methods
“Brain injury was determined by neurological deficit scores, neuroscore (28-point), corner test and gait analyses, at 24 and 72 h after stroke”
Conduct 28-point neuroscore evaluation to assess neurological deficits
Note: Part of comprehensive neurological assessment battery
“Brain injury was determined by neurological deficit scores, neuroscore (28-point), corner test and gait analyses, at 24 and 72 h after stroke”
Conduct corner test to assess neurological deficits and behavioral changes
Note: Part of comprehensive neurological assessment battery
“Brain injury was determined by neurological deficit scores, neuroscore (28-point), corner test and gait analyses, at 24 and 72 h after stroke”
Analyze gait to assess motor function and neurological deficits
Note: Part of comprehensive neurological assessment battery
“Brain injury was determined by neurological deficit scores, neuroscore (28-point), corner test and gait analyses, at 24 and 72 h after stroke”
Detect glutathione peroxidase 4 (GPx4), ACSL4 and cyclooxygenase 2 (COX2) protein levels
Note: Molecular analysis of ferroptosis-related proteins
“glutathione peroxidase 4 (GPx4), ACSL4 and cyclooxygenase 2 (COX2) were detected by Western blot”
Use transmission electron microscope to observe changes to mitochondria
Note: Ultrastructural analysis of mitochondrial damage
“changes to mitochondria were observed by a transmission electron microscope”
Use kit to determine iron levels as indicator of ferroptosis
Note: Iron accumulation is a hallmark of ferroptosis
“A kit was used to determine iron levels and lipid peroxide indicators”
Determine glutathione peroxidase (GPx), reduced glutathione (GSH), total glutathione/oxidized glutathione (GSH/GSSG), lipid peroxidation, reactive oxygen species, superoxide and malonaldehyde
Note: Multiple oxidative stress indicators measured using kit
“A kit was used to determine iron levels and lipid peroxide indicators, such as glutathione peroxidase (GPx), reduced glutathione (GSH), total glutathione/oxidized glutathione (GSH/GSSG), lipid peroxidation, reactive oxygen species, superoxide and malonaldehyde”
Determine brain infarct volume using 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining
Note: TTC staining performed at final timepoint
“Brain infarct volume was determined by 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining at 72 h after stroke”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
Assessment of brain injury using neurological deficit scores, neuroscore, corner test, and gait analysis following transient cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in mice
Objective
Assessment of brain injury using neurological deficit scores, neuroscore, corner test, and gait analysis following transient cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in mice
Subjects
From papermouse • Not specified • unknown • Not specified • Not specified
Cohort notes
From paperNone specified
Establish transient cerebral ischemia model (Not specified)
Administer Rosiglitazone (1 hour before MCAO)
Administer Liproxstatin-1 (Immediately after MCAO)
Perform neurological deficit scoring (At 24 and 72 hours post-stroke)
Neurological deficit scores
From paperNot specified
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
28-point neuroscore
From paperNot specified
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Corner test performance
From paperNot specified
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Gait analysis results
From paperNot specified
Artifact type
Longitudinal gait metrics and per-animal performance tables
Comparison focus
Compare recovery trajectory across post-injury timepoints and treatment conditions
Neurological deficit scores
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
28-point neuroscore
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Corner test performance
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Gait analysis results
From paperRaw 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
Final reported form
Group comparisons of gait indices, stride metrics, or recovery curves
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
Preprocessing / cleaning
Not specified
Scoring or quantification
Quantify the primary readouts for this experiment: Neurological deficit scores; 28-point neuroscore; Corner test performance; Gait analysis results.
Normalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Neurological deficit scores, 28-point neuroscore, Corner test performance, Gait analysis results.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Junmin Chen et al. (2021). Inhibition of Acyl-CoA Synthetase Long-Chain Family Member 4 Facilitates Neurological Recovery After Stroke by Regulation Ferroptosis. Frontiers in Cellular Neuroscience
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