Autophagy Promotes Peripheral Nerve Regeneration and Motor Recovery Following Sciatic Nerve Crush Injury in Rats methods
Aim. Evidence-backed execution summary for Autophagy Promotes Peripheral Nerve Regeneration and Motor Recovery Following Sciatic Nerve Crush Injury in Rats methods from Autophagy Promotes Peripheral Nerve Regeneration and Motor Recovery Following Sciatic Nerve Crush Injury in Rats.
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This experiment, in seven questions
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Drug Treatment
reagent used in the protocol.
- Use
- Animals were divided into four groups: sham + vehicle (v), crush + v, crush + rapamycin (rapa), and crush +3-methyladenine (3-MA). Rapamycin and 3-MA were obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 0.5 % dimethyl sulfoxide (DMSO). Animals received daily intraperitoneal injections of...
Immunocytochemistry
reagent used in the protocol.
- Use
- Transverse frozen sections (10-µm thickness) of sciatic nerve were dried, mounted onto slides, and washed in 0.1 M PBS and then blocked in 10 % goat serum and 3 % bovine serum albumin (BSA) for 1 h at 37 °C. After two washes with PBS, the slides were incubated with primary antibodi...
Western blot Analysis
reagent used in the protocol.
- Use
- LC3-II protein expression at the lesion site following sciatic NCI was analyzed by Western blotting. Nerve tissue samples were homogenized in tissue lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 % Triton X-100; 25 mM NaPPi; 80 mM β-glycerophosphate; 2 mM EDTA; 0.2...
TUNEL
reagent used in the protocol.
- Use
- DNA fragmentation resulting from cell death was detected in transverse frozen sections (10 µm) of sciatic nerves at the lesion site by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining using the TUNEL apoptosis assay kit (Beyotime Institute of Biotechnology) according to the man...
Behavioral Analysis
reagent used in the protocol.
- Use
- Experimenters performing the behavioral tests were blinded to the drug treatment conditions. Motor function was determined by stand time and footprint intensity using the CatWalk system (Noldus Inc., Wageningen, Netherlands). Animals crossed a walkway with an illuminated glass floor, and a GP-3360 high-speed video c...
Statistical Analysis
Data is presented as the mean ± SEM. Comparisons within groups were made by one-way analysis of variance (ANOVA), and between-groups comparisons were made by one-way ANOVA and, if significant, were followed by discrete comparisons using a post hoc test with a Bonferroni correction for multiple compari...
- Use
- Data is presented as the mean ± SEM. Comparisons within groups were made by one-way analysis of variance (ANOVA), and between-groups comparisons were made by one-way ANOVA and, if significant, were followed by discrete comparisons using a post hoc test with a Bonferroni correction for multiple compari...
Immunocytochemistry
Transverse frozen sections (10-µm thickness) of sciatic nerve were dried, mounted onto slides, and washed in 0.1 M PBS and then blocked in 10 % goat serum and 3 % bovine serum albumin (BSA) for 1 h at 37 °C. After two washes with PBS, the slides were incubated with primary antibodi...
- Use
- Transverse frozen sections (10-µm thickness) of sciatic nerve were dried, mounted onto slides, and washed in 0.1 M PBS and then blocked in 10 % goat serum and 3 % bovine serum albumin (BSA) for 1 h at 37 °C. After two washes with PBS, the slides were incubated with primary antibodi...
Western blot Analysis
LC3-II protein expression at the lesion site following sciatic NCI was analyzed by Western blotting. Nerve tissue samples were homogenized in tissue lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 % Triton X-100; 25 mM NaPPi; 80 mM β-glycerophosphate; 2 mM EDTA; 0.2...
- Use
- LC3-II protein expression at the lesion site following sciatic NCI was analyzed by Western blotting. Nerve tissue samples were homogenized in tissue lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 % Triton X-100; 25 mM NaPPi; 80 mM β-glycerophosphate; 2 mM EDTA; 0.2...
Electron Microscopy
Sciatic nerve segments were removed 1 week after NCI and fixed with 2.5 % glutaraldehyde overnight at 4 °C. The segments were cut with a sharp razor blade into serial 1-mm transverse sections around the site of injury, which were post-fixed in 1 % osmium tetroxide for 1 h at 4 ...
- Use
- Sciatic nerve segments were removed 1 week after NCI and fixed with 2.5 % glutaraldehyde overnight at 4 °C. The segments were cut with a sharp razor blade into serial 1-mm transverse sections around the site of injury, which were post-fixed in 1 % osmium tetroxide for 1 h at 4 ...
Behavioral Analysis
Experimenters performing the behavioral tests were blinded to the drug treatment conditions. Motor function was determined by stand time and footprint intensity using the CatWalk system (Noldus Inc., Wageningen, Netherlands). Animals crossed a walkway with an illuminated glass floor, and a GP-3360 high-speed video c...
- Use
- Experimenters performing the behavioral tests were blinded to the drug treatment conditions. Motor function was determined by stand time and footprint intensity using the CatWalk system (Noldus Inc., Wageningen, Netherlands). Animals crossed a walkway with an illuminated glass floor, and a GP-3360 high-speed video c...
Autophagy Promotes Axonal Regeneration and SC Remyelination
MBP is a major constituent of the myelin sheath produced by SCs in the peripheral nervous system, while the expression of NF-200-an axon-specific intermediate filament-is critical for axon stabilization during their maturation. To investigate the effects of autophagy on nerve regeneration, the expression...
- Use
- MBP is a major constituent of the myelin sheath produced by SCs in the peripheral nervous system, while the expression of NF-200-an axon-specific intermediate filament-is critical for axon stabilization during their maturation. To investigate the effects of autophagy on nerve regeneration, the expression...
Statistical Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data is presented as the mean ± SEM. Comparisons within groups were made by one-way analysis of variance (ANOVA), and between-groups comparisons were made by one-way ANOVA and, if significant, were followed by discrete comparisons using a post hoc test with a Bonferroni correction for multiple compari...
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Drug Treatment
Animals were divided into four groups: sham + vehicle (v), crush + v, crush + rapamycin (rapa), and crush +3-methyladenine (3-MA). Rapamycin and 3-MA were obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 0.5 % dimethyl sulfoxide (DMSO). Animals received daily intraperitoneal injections of rapamycin at a dose of 1 mg/kg (crush + rapa group), 3-MA at a dose of 50 mg/kg (crush +3-MA group), or DMSO (crush + v group) for 5 days after the surgery. Sham-operated rats (sham + v group), in which the sciatic nerve was exposed but not subjected to crush injury, received daily intraperitoneal injections of an equal volume (1 mL) 0.5 % DMSO for 5 days. Drug dosages were selected based on data from previous studies and preliminary experiments (Maeda et al.; Chen et al.; Sekiguchi et al. ).
Immunocytochemistry
Transverse frozen sections (10-µm thickness) of sciatic nerve were dried, mounted onto slides, and washed in 0.1 M PBS and then blocked in 10 % goat serum and 3 % bovine serum albumin (BSA) for 1 h at 37 °C. After two washes with PBS, the slides were incubated with primary antibodies against MBP (1:1000; Sigma), NF-200 (1:500; Sigma), or LC3B-II (1: 500; Cell Signaling Technology, Danvers, MA, USA) at 4 °C for 12 h, followed by repeated washes with PBS, and then incubated with Alexa 488- or Cy3-conjugated secondary antibodies (1:1000; Invitrogen, Shanghai, China) for 2 h at room temperature. Sections were visualized under an epifluorescence microscope (Leica, DM6000B, Wetzlar, Germany) and imaged at × 400 magnification.
Western blot Analysis
LC3-II protein expression at the lesion site following sciatic NCI was analyzed by Western blotting. Nerve tissue samples were homogenized in tissue lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 % Triton X-100; 25 mM NaPPi; 80 mM β-glycerophosphate; 2 mM EDTA; 0.2 mM Na3VO4 and protease inhibitor cocktail) and centrifuged at 12,000 g for 15 min at 4 °C. The protein concentration in the supernatant was measured with a bicinchoninic assay kit (Beyotime Institute of Biotechnology, Shanghai, China), and the supernatant was mixed with sample buffer (50 mM Tris-HCl, 10 % sodium dodecyl sulfate, 10 % glycerol, 10 % 2-mercaptoethanol, and 2 mg/ml bromophenol blue) at a 1:1 ratio and boiled for 5 min. A total of 20 µg of each sample was separated on a 15 % acrylamide gel and transfer...
Measurement outputs
What raw and processed outputs should exist?
Data is presented as the mean ± SEM. Comparisons within groups were made by one-way analysis of variance (ANOVA), and between-groups comparisons were made by one-...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Sciatic nerves were dissected and harvested 1 and 6 weeks post-injury ( n = 6 for each time point and group). Animals were decapitated and perfused via the left...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
LC3-II protein expression at the lesion site following sciatic NCI was analyzed by Western blotting. Nerve tissue samples were homogenized in tissue lysis buffer (20 mM Tri...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Experimenters performing the behavioral tests were blinded to the drug treatment conditions. Motor function was determined by stand time and footprint intensity using the CatWal...
- 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
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
Data is presented as the mean ± SEM.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Data is presented as the mean ± SEM. Comparisons within groups were made by one-way analysis of variance (ANOVA), and between-groups comparisons were made by one-...; Sciatic nerves were dissected and harvested 1 and 6 weeks post-injury ( n = 6 for each time point and group). Animals were decapitated and perfused via the left...; LC3-II protein expression at the lesion site following sciatic NCI was analyzed by Western blotting. Nerve tissue samples were homogenized in tissue lysis buffer (20 mM Tri...; Experimenters performing the behavioral tests were blinded to the drug treatment conditions. Motor function was determined by stand time and footprint intensity using the CatWal....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Data is presented as the mean ± SEM. Comparisons within groups were made by one-way analysis of variance (ANOVA), and between-groups comparisons were made by one-...; The lesion site of the sciatic nerve was examined 6 weeks post-injury by electron microscopy (Fig. ). Axon number and G-ratio-which is the ratio of inner to outer dia...
from paperReporting output
Report representative outputs alongside summary comparisons for Data is presented as the mean ± SEM. Comparisons within groups were made by one-way analysis of variance (ANOVA), and between-groups comparisons were made by one-..., Sciatic nerves were dissected and harvested 1 and 6 weeks post-injury ( n = 6 for each time point and group). Animals were decapitated and perfused via the left..., LC3-II protein expression at the lesion site following sciatic NCI was analyzed by Western blotting. Nerve tissue samples were homogenized in tissue lysis buffer (20 mM Tri..., Experimenters performing the behavioral tests were blinded to the drug treatment conditions. Motor function was determined by stand time and footprint intensity using the CatWal....
inferred from protocolStructured statistical methods
Data is presented as the mean ± SEM. Comparisons within groups were made by one-way analysis of variance (ANOVA), and between-groups comparisons were made by one-...; The lesion site of the sciatic nerve was examined 6 weeks post-injury by electron microscopy (Fig. ). Axon number and G-ratio-which is the ratio of inner to outer dia...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (3)
Animals were divided into four groups: sham + vehicle (v), crush + v, crush + rapamycin (rapa), and crush +3-methyladenine (3-MA). Rapamycin and 3-MA were obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 0.5 % dimethyl sulfoxide (DMSO). Animals received daily intraperitoneal injections of rapamycin at a dose of 1 mg/kg (crush + rapa group), 3-MA at a dose of 50 mg/kg (crush +3-MA group), or DMSO (crush + v group) for 5 days after the surgery. Sham-operated rats (sham + v group), in which the sciatic nerve was exposed but not subjected to crush injury, received daily intraperitoneal injections of an equal volume (1 mL) 0.5 % DMSO for 5 days. Drug dosages were selected based on data from previous studies and preliminary experiments (Maeda et al.; Chen et al.; Sekiguchi et al. ).
Transverse frozen sections (10-µm thickness) of sciatic nerve were dried, mounted onto slides, and washed in 0.1 M PBS and then blocked in 10 % goat serum and 3 % bovine serum albumin (BSA) for 1 h at 37 °C. After two washes with PBS, the slides were incubated with primary antibodies against MBP (1:1000; Sigma), NF-200 (1:500; Sigma), or LC3B-II (1: 500; Cell Signaling Technology, Danvers, MA, USA) at 4 °C for 12 h, followed by repeated washes with PBS, and then incubated with Alexa 488- or Cy3-conjugated secondary antibodies (1:1000; Invitrogen, Shanghai, China) for 2 h at room temperature. Sections were visualized under an epifluorescence microscope (Leica, DM6000B, Wetzlar, Germany) and imaged at × 400 magnification.
LC3-II protein expression at the lesion site following sciatic NCI was analyzed by Western blotting. Nerve tissue samples were homogenized in tissue lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 % Triton X-100; 25 mM NaPPi; 80 mM β-glycerophosphate; 2 mM EDTA; 0.2 mM Na3VO4 and protease inhibitor cocktail) and centrifuged at 12,000 g for 15 min at 4 °C. The protein concentration in the supernatant was measured with a bicinchoninic assay kit (Beyotime Institute of Biotechnology, Shanghai, China), and the supernatant was mixed with sample buffer (50 mM Tris-HCl, 10 % sodium dodecyl sulfate, 10 % glycerol, 10 % 2-mercaptoethanol, and 2 mg/ml bromophenol blue) at a 1:1 ratio and boiled for 5 min. A total of 20 µg of each sample was separated on a 15 % acrylamide gel and transferred to a nitrocellulose membrane (BioScience, Shanghai, China) by electroblotting (Bio-Rad, Hercules, California, USA) at 120 V for 1.5 h followed by 70 V for 0.5 h at 4 °C. Membranes were blocked with 3 % BSA and incubated with rabbit anti-LC3B-II antibody (1:000)...
Machine-readable layer
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