Inhibition of Acyl-CoA Synthetase Long-Chain Family Member 4 Facilitates Neurological Recovery After Stroke by Regulation Ferroptosis methods
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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 a...
Materials and Methods
Male C57BL/6 mice (age 8-10 weeks, weight 23-25 g, RRID: IMSR_CRL:27 ) of specific-pathogen-free grade were purchased from the Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animals were kept on a 12-h light/dark cycle with controlled humidity (60% ± 5%) and temperature (22°C ± 3°C) and had free access to food and water. All animals were allowed to acclimatize to the new surroundings for at least 3 days before use in experiments. Animals were assigned randomly using a random number table. All assessments were conducted by investigators who were blinded to experimental group assignment. All experimental procedures were approved by the Committee of Experimental Animals of Hebei Medical University (Shijiazhuang, China) and conducted in accordance with the National Institutes of Health guidelines. Sample sizes were not estimated pri...
Transient Cerebral Ischemia by Middle Cerebral Artery Occlusion
Mice were fixed and anesthetized by intraperitoneal (i.p.) injection of 1% pentobarbital (100 mg/kg). Transient focal cerebral ischemia was produced in the right middle cerebral artery occlusion (MCAO) using the intraluminal filament technique. The monofilament was withdrawn after 1 h to restore blood flow. The right carotid artery was exposed to dissect the external carotid arteries (ECA) and the internal carotid arteries (ICA), blocking the ECA at the level of the middle cerebral artery (MCA) branch. A monofilament nylon suture (Yushun Biotech, Guangzhou, China) with a rounded tip was inserted through the ICA. When slight resistance was felt, threading was stopped and fixed. After 1 h, the filament was gently removed for reperfusion. The mock-operated control mice underwent the same steps except for inserting the filament. Before, during and after MCAO, the local cerebral blood flow...
Inhibitor Treatment and Experimental Design
Liproxstatin-1 (Lip, MedChemExpress Cat#HY-10108), a ferroptosis inhibitor, was administered intranasally immediately after MCAO at a concentration of 10 mg/kg ( ). Rosiglitazone (ROSI, Selleck Cat#S2556) is used to inhibit ACSL4, a classic peroxisome proliferator-activated receptor-γ agonist. In the current study, it which was administered intravenously 1 h before MCAO at a concentration of 0.4 mg/kg. The mice were randomly divided using a random number table generated by SPSS software. In preliminary experiments, mice were randomly assigned into two groups: the sham group (mice receiving a mock operation) and the MCAO group (mice received MCAO). In subsequent experiments, mice were randomly assigned into four groups: sham group, MCAO group, sham + Lip group (mice receiving mock treatment and intranasally liproxstatin-1) and MCAO + Lip group (mice receiving MCAO and intranasally...
Neurobehavioral Tests
CatWalk XT (v10.6, Noldus Information Technology), a gait analysis system, was used to measure footfalls and motor performance in ischemic mice as described in. Each mouse walked freely through a corridor on a glass sidewalk that was illuminated from below. A camera was used to collect the movements of all four paws at a rate of 100 frames/sec for 20 s; CatWalk XT software was then used for analysis. Walking at a steady speed (without stopping, grooming, or rearing) was considered a successful walking test. Prior to the MCAO, the mice in each group were continuously trained for 3 days. For the test, mice were placed on a glass sidewalk with a speed of 8 cm/s or the maximum speed at which mice could maintain optimally coordinated locomotion. Background images were recorded prior to each test day. Each mouse was performed three times, with an interval of more than 15 min each time. Ins...
Measurement of Infarction Volume
2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed to evaluate the infarct volume at 72 h after reperfusion. Brain tissue was sliced into seven coronal sections (1mm thick), stained with a 2% solution of TTC. Percentage hemisphereisphere lesion volume = {[total infarct volume - (volume of intact ipsilateral hemisphere - volume of intact contralateral hemisphere)]/contralateral hemisphere volume}.
Western Blot
Cortical tissue was extracted from the peri-infarct area 24 h after MCAO and from the corresponding area of the mock-operated mice. Protein from cortical tissue was extracted using a total protein extraction kit (Applygen Cat#P1250) with a 2% protease inhibitor cocktail (Sigma Cat# P8340) and a 1% phosphatase inhibitor (Applygen Cat#P1260), according to the description in, and the entire extraction process was performed on ice or at 4°C. The protein concentration of the tissue sample was measured using a bicinchoninic acid (BCA) protein assay reagent kit (Thermo Scientific, United States). An equivalent amount of protein (50 µg) was separated by a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Roche, United States). The PVDF membranes were then blocked with 5% non-fat milk powder at room tempe...
Transmission Electron Microscopy
Mice were deeply anesthetized and perfused with precooled phosphate-buffered saline (PBS, pH 7.4) and 2.5% glutaraldehyde (Servicebio Cat#G1102). A 1-mm-cube tissue block was obtained from the peri-infarct area 24 h after MCAO and from the corresponding area of the mock-operated mice. The tissue blocks were then steeped in the 2.5% glutaraldehyde at 4°C for 2-4 h. The samples were post-fixed with 1% osmium tetroxide in a 0.1 M phosphate buffer (PB, pH 7.4) at room temperature for 2 h, dehydrated in a graded ethyl alcohol series, and embedded in SPI-Pon 812 epoxy resin (SPI Cat#90529-77-4) overnight at 37°C. The polymerization was performed at 60°C for 48 h. Ultrathin sections (60-80 nm) were cut using a Leica UC7 ultramicrotome, stained with 2% uranyl acetate and lead citrate for 15 min, and viewed under an HT7700 transmission electron microscope (TEM, HITAC...
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
- Reported as
- 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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Quantify the primary readouts for this experiment: Gait recovery and locomotor function; Protein expression by western blot.
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inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function, Protein expression by western blot.
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Evidence quotes (8)
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.
Male C57BL/6 mice (age 8-10 weeks, weight 23-25 g, RRID: IMSR_CRL:27 ) of specific-pathogen-free grade were purchased from the Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animals were kept on a 12-h light/dark cycle with controlled humidity (60% ± 5%) and temperature (22°C ± 3°C) and had free access to food and water. All animals were allowed to acclimatize to the new surroundings for at least 3 days before use in experiments. Animals were assigned randomly using a random number table. All assessments were conducted by investigators who were blinded to experimental group assignment. All experimental procedures were approved by the Committee of Experimental Animals of Hebei Medical University (Shijiazhuang, China) and conducted in accordance with the National Institutes of Health guidelines. Sample sizes were not estimated prior to conducting experiments. Rather, animals were allocated into each group until sample sizes were similar to that of other MCAO studies. The mice were randomly divided using a random number table generated by SPSS software (v. 21.0; SPSS, RRID:SCR_002865 ) ( ).
Mice were fixed and anesthetized by intraperitoneal (i.p.) injection of 1% pentobarbital (100 mg/kg). Transient focal cerebral ischemia was produced in the right middle cerebral artery occlusion (MCAO) using the intraluminal filament technique. The monofilament was withdrawn after 1 h to restore blood flow. The right carotid artery was exposed to dissect the external carotid arteries (ECA) and the internal carotid arteries (ICA), blocking the ECA at the level of the middle cerebral artery (MCA) branch. A monofilament nylon suture (Yushun Biotech, Guangzhou, China) with a rounded tip was inserted through the ICA. When slight resistance was felt, threading was stopped and fixed. After 1 h, the filament was gently removed for reperfusion. The mock-operated control mice underwent the same steps except for inserting the filament. Before, during and after MCAO, the local cerebral blood flow was monitored by laser Doppler flowmeter (Moor VMS-LDF, Moor Instruments Ltd., Axminster, Devon, United Kingdom), and the MCA was occluded based on a criterion of a < 25% baseline blood flow remaining after MCAO. A constant temperature heating pad at 37°C was used during and after the operatio...
Liproxstatin-1 (Lip, MedChemExpress Cat#HY-10108), a ferroptosis inhibitor, was administered intranasally immediately after MCAO at a concentration of 10 mg/kg ( ). Rosiglitazone (ROSI, Selleck Cat#S2556) is used to inhibit ACSL4, a classic peroxisome proliferator-activated receptor-γ agonist. In the current study, it which was administered intravenously 1 h before MCAO at a concentration of 0.4 mg/kg. The mice were randomly divided using a random number table generated by SPSS software. In preliminary experiments, mice were randomly assigned into two groups: the sham group (mice receiving a mock operation) and the MCAO group (mice received MCAO). In subsequent experiments, mice were randomly assigned into four groups: sham group, MCAO group, sham + Lip group (mice receiving mock treatment and intranasally liproxstatin-1) and MCAO + Lip group (mice receiving MCAO and intranasally liproxstatin-1). Additionally, mice were randomly assigned into four groups: sham, MCAO, sham + ROSI (mice receiving a mock operation and intravenously rosiglitazone) and MCAO + ROSI groups (mice receiving MCAO and intravenously rosiglitazone). According to the experimental design, the mice were qu...
CatWalk XT (v10.6, Noldus Information Technology), a gait analysis system, was used to measure footfalls and motor performance in ischemic mice as described in. Each mouse walked freely through a corridor on a glass sidewalk that was illuminated from below. A camera was used to collect the movements of all four paws at a rate of 100 frames/sec for 20 s; CatWalk XT software was then used for analysis. Walking at a steady speed (without stopping, grooming, or rearing) was considered a successful walking test. Prior to the MCAO, the mice in each group were continuously trained for 3 days. For the test, mice were placed on a glass sidewalk with a speed of 8 cm/s or the maximum speed at which mice could maintain optimally coordinated locomotion. Background images were recorded prior to each test day. Each mouse was performed three times, with an interval of more than 15 min each time. Instantaneous running speed (mm/s), stride-length of the left front limb (mm) and the print area of the left front limb (mm 2 ) were selected for gait analysis.
2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed to evaluate the infarct volume at 72 h after reperfusion. Brain tissue was sliced into seven coronal sections (1mm thick), stained with a 2% solution of TTC. Percentage hemisphereisphere lesion volume = {[total infarct volume - (volume of intact ipsilateral hemisphere - volume of intact contralateral hemisphere)]/contralateral hemisphere volume}.
Cortical tissue was extracted from the peri-infarct area 24 h after MCAO and from the corresponding area of the mock-operated mice. Protein from cortical tissue was extracted using a total protein extraction kit (Applygen Cat#P1250) with a 2% protease inhibitor cocktail (Sigma Cat# P8340) and a 1% phosphatase inhibitor (Applygen Cat#P1260), according to the description in, and the entire extraction process was performed on ice or at 4°C. The protein concentration of the tissue sample was measured using a bicinchoninic acid (BCA) protein assay reagent kit (Thermo Scientific, United States). An equivalent amount of protein (50 µg) was separated by a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Roche, United States). The PVDF membranes were then blocked with 5% non-fat milk powder at room temperature for 1 h and incubated with the primary antibodies overnight at 4°C. Primary antibodies included GPx4 (1:5000, Abcam Cat#ab125066, RRID: AB_10973901 ), ACSL4 (1:1000, Abcam Cat# ab155282, RRID: AB_2714020 ), COX2 (1:800, Abcam Cat#ab62331, RRID: AB_942121 ) and GAPDH (1:10000, Bioworld Te...
Mice were deeply anesthetized and perfused with precooled phosphate-buffered saline (PBS, pH 7.4) and 2.5% glutaraldehyde (Servicebio Cat#G1102). A 1-mm-cube tissue block was obtained from the peri-infarct area 24 h after MCAO and from the corresponding area of the mock-operated mice. The tissue blocks were then steeped in the 2.5% glutaraldehyde at 4°C for 2-4 h. The samples were post-fixed with 1% osmium tetroxide in a 0.1 M phosphate buffer (PB, pH 7.4) at room temperature for 2 h, dehydrated in a graded ethyl alcohol series, and embedded in SPI-Pon 812 epoxy resin (SPI Cat#90529-77-4) overnight at 37°C. The polymerization was performed at 60°C for 48 h. Ultrathin sections (60-80 nm) were cut using a Leica UC7 ultramicrotome, stained with 2% uranyl acetate and lead citrate for 15 min, and viewed under an HT7700 transmission electron microscope (TEM, HITACHI, Japan).
Machine-readable layer
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"text": "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 a..."
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"headline": "Inhibition of Acyl-CoA Synthetase Long-Chain Family Member 4 Facilitates Neurological Recovery After Stroke by Regulation Ferroptosis",
"datePublished": "2021",
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"identifier": "10.3389/fncel.2021.632354"
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