Source Paper
Inhibition of Acyl-CoA Synthetase Long-Chain Family Member 4 Facilitates Neurological Recovery After Stroke by Regulation Ferroptosis
Junmin Chen, et al.
Source Paper
Junmin Chen, 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: Establish a transient cerebral ischemia model by middle cerebral artery occlusion (MCAO) in mice and evaluate ferroptosis inhibition and ACSL4 inhibition on brain injury and oxidative stress markers
This is a Middle Cerebral Artery Occlusion protocol using mouse as the model organism. The procedure involves 9 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
~3 day study window | ~243 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 establish transient cerebral ischemia model
Note: Specific surgical technique and duration of occlusion not detailed in provided text
“A transient cerebral ischemia model was established for mice by middle cerebral artery occlusion (MCAO)”
Administer rosiglitazone intravenously as an ACSL4 inhibitor 1 hour before MCAO at 0.4 mg/kg concentration
Note: Timing is critical - must be administered 1 hour prior to occlusion
“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 at 10 mg/kg concentration
Note: Intranasal administration route; timing is immediately post-MCAO
“Following MCAO, a ferroptosis inhibitor, liproxstatin-1, was administered intranasally immediately at a concentration of 10 mg/kg”
Collect brain tissue samples for detection of glutathione peroxidase 4 (GPx4), ACSL4, and cyclooxygenase 2 (COX2) by Western blot
Note: Timing of tissue collection not specified in provided text
“glutathione peroxidase 4 (GPx4), ACSL4 and cyclooxygenase 2 (COX2) were detected by Western blot”
Prepare brain tissue samples to observe changes to mitochondria by transmission electron microscope
Note: Specific preparation methods not detailed in provided text
“changes to mitochondria were observed by a transmission electron microscope”
Use kit to determine iron levels and lipid peroxide indicators including glutathione peroxidase (GPx), reduced glutathione (GSH), total glutathione/oxidized glutathione (GSH/GSSG), lipid peroxidation, reactive oxygen species, superoxide and malonaldehyde
Note: Specific kit manufacturer and catalog numbers not provided
“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”
Perform neurological deficit scoring using 28-point neuroscore, corner test, and gait analyses at 24 hours after stroke
Note: Multiple behavioral assessments performed at this timepoint
“Brain injury was determined by neurological deficit scores, neuroscore (28-point), corner test and gait analyses, at 24 and 72 h after stroke”
Perform neurological deficit scoring using 28-point neuroscore, corner test, and gait analyses at 72 hours after stroke
Note: Multiple behavioral assessments performed at this timepoint
“Brain injury was determined by neurological deficit scores, neuroscore (28-point), corner test and gait analyses, at 24 and 72 h after stroke”
Perform 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining to determine brain infarct volume at 72 hours after stroke
Note: TTC staining performed at 72 hour timepoint only
“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.
Establish a transient cerebral ischemia model by middle cerebral artery occlusion (MCAO) in mice and evaluate ferroptosis inhibition and ACSL4 inhibition on brain injury and oxidative stress markers
Objective
Establish a transient cerebral ischemia model by middle cerebral artery occlusion (MCAO) in mice and evaluate ferroptosis inhibition and ACSL4 inhibition on brain injury and oxidative stress markers
Subjects
From papermouse • Not specified • unknown • Not specified • Not specified
Cohort notes
From paperNot specified
Establish transient cerebral ischemia model (Not specified)
Administer rosiglitazone (1 hour before MCAO)
Administer liproxstatin-1 (Immediately after MCAO)
Collect tissue samples for Western blot analysis (Not specified)
Glutathione peroxidase 4 (GPx4) protein levels
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
ACSL4 protein levels
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
Cyclooxygenase 2 (COX2) protein levels
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
Mitochondrial morphology and changes
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
Glutathione peroxidase 4 (GPx4) protein levels
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
ACSL4 protein levels
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
Cyclooxygenase 2 (COX2) protein levels
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
Mitochondrial morphology and changes
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
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: Glutathione peroxidase 4 (GPx4) protein levels; ACSL4 protein levels; Cyclooxygenase 2 (COX2) protein levels; Mitochondrial morphology and changes.
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 Glutathione peroxidase 4 (GPx4) protein levels, ACSL4 protein levels, Cyclooxygenase 2 (COX2) protein levels, Mitochondrial morphology and changes.
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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