Ablation of ferroptosis regulator glutathione peroxidase 4 in forebrain neurons promotes cognitive impairment and neurodegeneration methods
Aim. Evidence-backed execution summary for Ablation of ferroptosis regulator glutathione peroxidase 4 in forebrain neurons promotes cognitive impairment and neurodegeneration methods from Ablation of ferroptosis regulator glutathione peroxidase 4 in forebrain neurons promotes cognitive impairment and neurodegeneration.
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mouse
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Materials and methods
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- Use
- Liproxstatin-1, a small-molecule ferroptosis inhibitor, was purchased from Selleckchem.com (Cat #-S7699). To evaluate the effect of ferroptosis inhibition on neurodegeneration, after being fed the vitamin E deficient diet for 6 weeks, Gpx4BIKO mice were treated with tamoxifen to ablate Gpx4, as described above. Two...
Results
reagent used in the protocol.
- Use
- We next tested the conditional ablation of Gpx4 in Gpx4BIKO mice. Gpx4BIKO mice (2-3 months of age) were treated with tamoxifen (TAM), and tissues from those mice were collected 2 weeks later for analysis. First, we used a PCR-based method to detect the recombined Gpx4 allele (rGpx4) in tissues from Gpx4BIKO m...
Materials and methods
All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committees of the University of Texas Health Science Center at San Antonio and the Audie Murphy Memorial Veterans Hospital, South Texas Veterans Health Care System.
- Use
- All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committees of the University of Texas Health Science Center at San Antonio and the Audie Murphy Memorial Veterans Hospital, South Texas Veterans Health Care System.
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Materials and methods
Liproxstatin-1, a small-molecule ferroptosis inhibitor, was purchased from Selleckchem.com (Cat #-S7699). To evaluate the effect of ferroptosis inhibition on neurodegeneration, after being fed the vitamin E deficient diet for 6 weeks, Gpx4BIKO mice were treated with tamoxifen to ablate Gpx4, as described above. Two days into tamoxifen treatment, liproxstatin-1, which was dissolved in DMSO and then diluted in PBS, was given to one half of the mice via i.p. injection every two days at a dose of 10 mg/kg, whereas the other half of mice received i.p. injection of the vehicle (1.5% DMSO in PBS) only.
Materials and methods
To induce Gpx4 ablation, tamoxifen (T5648, Sigma) was dissolved in corn oil (10 mg/ml) and administered by intraperitoneal (i.p.) injection to Gpx4BIKO mice at 60 mg/kg once per day for 5 days. The same treatment also was applied to control Gpx4(f/f) mice.
Materials and methods
The vitamin E deficient diet, an AIN-93G-modified rodent diet that lacks vitamin E, was formulated and manufactured by Bio-Serv (Flemington, NJ). To determine the effect of vitamin E deficiency on neurodegeneration, Gpx4BIKO mice and control Gpx4(f/f) mice were fed the vitamin E deficient diet starting at the age of 1 month. Tamoxifen was administered 6 weeks later to ablate Gpx4 in Gpx4BIKO mice.
Results
We next tested the conditional ablation of Gpx4 in Gpx4BIKO mice. Gpx4BIKO mice (2-3 months of age) were treated with tamoxifen (TAM), and tissues from those mice were collected 2 weeks later for analysis. First, we used a PCR-based method to detect the recombined Gpx4 allele (rGpx4) in tissues from Gpx4BIKO mice ( A). As B shows, rGpx4 was present in cerebral cortex and hippocampus tissues of TAM-treated Gpx4BIKO mice but not in spinal cord or liver tissues. Notably, rGpx4 was not detectable in cerebral cortex and hippocampus tissues from Gpx4BIKO mice without TAM treatment or in tissues from Gpx4(f/f) mice. We next compared levels of Gpx4 protein between TAM-treated Gpx4BIKO mice and control Gpx4(f/f) mice by Western blots. As C shows, TAM-treated Gpx4BIKO mice had a decreased level of Gpx4 protein in cortex and hippocampus tissues compared with control Gpx4(f/f) mice, and the...
Measurement outputs
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A PCR-based method was used to detect Cre-mediated recombination of the floxed Gpx4 allele in genomic DNA isolated from tissues. PCR reactions were performed using primers (P1,...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
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- Summary statistics and between-group or across-timepoint comparisons
We next tested the conditional ablation of Gpx4 in Gpx4BIKO mice. Gpx4BIKO mice (2-3 months of age) were treated with tamoxifen (TAM), and tissues from those mice were col...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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inferred from protocolPreprocessing / cleaning
We next tested the conditional ablation of Gpx4 in Gpx4BIKO mice.
from paperScoring or quantification
Quantify the primary readouts for this experiment: A PCR-based method was used to detect Cre-mediated recombination of the floxed Gpx4 allele in genomic DNA isolated from tissues. PCR reactions were performed using primers (P1,...; We next tested the conditional ablation of Gpx4 in Gpx4BIKO mice. Gpx4BIKO mice (2-3 months of age) were treated with tamoxifen (TAM), and tissues from those mice were col....
from paperStatistical comparison
We next tested the conditional ablation of Gpx4 in Gpx4BIKO mice. Gpx4BIKO mice (2-3 months of age) were treated with tamoxifen (TAM), and tissues from those mice were col...
from paperReporting output
Report representative outputs alongside summary comparisons for A PCR-based method was used to detect Cre-mediated recombination of the floxed Gpx4 allele in genomic DNA isolated from tissues. PCR reactions were performed using primers (P1,..., We next tested the conditional ablation of Gpx4 in Gpx4BIKO mice. Gpx4BIKO mice (2-3 months of age) were treated with tamoxifen (TAM), and tissues from those mice were col....
inferred from protocolStructured statistical methods
We next tested the conditional ablation of Gpx4 in Gpx4BIKO mice. Gpx4BIKO mice (2-3 months of age) were treated with tamoxifen (TAM), and tissues from those mice were col...
source structuredSource and audit
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Evidence quotes (4)
Liproxstatin-1, a small-molecule ferroptosis inhibitor, was purchased from Selleckchem.com (Cat #-S7699). To evaluate the effect of ferroptosis inhibition on neurodegeneration, after being fed the vitamin E deficient diet for 6 weeks, Gpx4BIKO mice were treated with tamoxifen to ablate Gpx4, as described above. Two days into tamoxifen treatment, liproxstatin-1, which was dissolved in DMSO and then diluted in PBS, was given to one half of the mice via i.p. injection every two days at a dose of 10 mg/kg, whereas the other half of mice received i.p. injection of the vehicle (1.5% DMSO in PBS) only.
To induce Gpx4 ablation, tamoxifen (T5648, Sigma) was dissolved in corn oil (10 mg/ml) and administered by intraperitoneal (i.p.) injection to Gpx4BIKO mice at 60 mg/kg once per day for 5 days. The same treatment also was applied to control Gpx4(f/f) mice.
The vitamin E deficient diet, an AIN-93G-modified rodent diet that lacks vitamin E, was formulated and manufactured by Bio-Serv (Flemington, NJ). To determine the effect of vitamin E deficiency on neurodegeneration, Gpx4BIKO mice and control Gpx4(f/f) mice were fed the vitamin E deficient diet starting at the age of 1 month. Tamoxifen was administered 6 weeks later to ablate Gpx4 in Gpx4BIKO mice.
We next tested the conditional ablation of Gpx4 in Gpx4BIKO mice. Gpx4BIKO mice (2-3 months of age) were treated with tamoxifen (TAM), and tissues from those mice were collected 2 weeks later for analysis. First, we used a PCR-based method to detect the recombined Gpx4 allele (rGpx4) in tissues from Gpx4BIKO mice ( A). As B shows, rGpx4 was present in cerebral cortex and hippocampus tissues of TAM-treated Gpx4BIKO mice but not in spinal cord or liver tissues. Notably, rGpx4 was not detectable in cerebral cortex and hippocampus tissues from Gpx4BIKO mice without TAM treatment or in tissues from Gpx4(f/f) mice. We next compared levels of Gpx4 protein between TAM-treated Gpx4BIKO mice and control Gpx4(f/f) mice by Western blots. As C shows, TAM-treated Gpx4BIKO mice had a decreased level of Gpx4 protein in cortex and hippocampus tissues compared with control Gpx4(f/f) mice, and the decreased level of Gpx4 protein was maintained at 15 weeks after TAM treatment. We also looked at the neuronal level of Gpx4 by immunofluorescence. Brain sections from TAM-treated Gpx4BIKO mice and control mice were stained with the anti-Gpx4 antibody. As D shows, hippocampal neurons from TAM-treat...
Machine-readable layer
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