Source Paper
Hai-cheng Huang, Li Chen, Hai-xing Zhang, Sheng-fa Li, Pei Liu et al.
Journal of Molecular Neuroscience • 2016
Abstract Autophagy maintains cellular homeostasis by stimulating the lysosomal degradation of cytoplasmic structures, including damaged organelles and dysfunctional proteins. The role of autophagy in the renewal and regeneration of injured peripheral nerves remains poorly understood. The current study investigated the role of autophagy in peripheral nerve regeneration and motor function recovery following sciatic nerve crush injury in rats by stimulating or suppressing autophagy and detecting the presence of autophagosomes and LC3-II expression by electron microscopy and Western blotting, respectively. Neurobehavioral function was tested by CatWalk gait analysis 1, 2, 3, and 6 weeks after injury, and the expression of neurofilament (NF)-200 and myelin basic protein (MBP) at the injury site was examined by immunocytochemistry. Apoptosis at the lesion site was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Treatment of injured rats with the autophagy inducer rapamycin increased the number of autophagosomes and LC3-II expression while reducing the number of apoptotic cells at the lesion; this was associated with an upregulation of MBP and NF-200 expression and increased motor function recovery as compared to sham-operated rats and those that were subjected to crush injury but untreated. The opposite effects were observed in rats treated with the autophagy inhibitor 3-methyladenine. These data indicate that the modulation of autophagy in peripheral nerve injury could be an effective pharmacological approach to promote nerve regeneration and reestablish motor function.
Objective: Induce sciatic nerve crush injury in rats, pharmacologically modulate autophagy with rapamycin or 3-methyladenine, and quantify motor recovery and tissue-level regeneration across behavioral, immunocytochemical, western blot, electron microscopy, and TUNEL assays.
This is a Sciatic nerve crush injury with autophagy modulation and motor recovery assessment protocol using rat as the model organism. The procedure involves 8 procedural steps, 6 equipment items, 9 materials. Extracted from a 2016 paper published in Journal of Molecular Neuroscience.
Model and subjects
rat • Sprague-Dawley • female • Adult • 180-220g
Study window
~6 week study window
Core workflow
Induce sciatic nerve crush injury • Dose rapamycin, 3-MA, or vehicle after surgery • Harvest and fix sciatic nerve tissue
Primary readouts
Key equipment and reagents
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Deeply anesthetize adult female Sprague-Dawley rats with sodium pentobarbital, expose the right sciatic nerve in the mid-thigh, and create crush injury by clamping it three times with forceps for 10 seconds each at 10-second intervals.
Note: Forceps-based crush injury model in the right sciatic nerve.
“rats were deeply anesthetized with sodium pentobarbital (50 mg/kg body weight by intraperitoneal injection), and the sciatic nerve in the right mid-thigh was exposed and clamped with a pair of forceps three times for 10 s each at 10-s intervals”
Prepare rapamycin and 3-methyladenine in 0.5% DMSO, then administer daily intraperitoneal injections for 5 days after surgery to the assigned experimental groups, with sham and vehicle groups receiving equal-volume DMSO control injections.
Note: Rapamycin 1 mg/kg; 3-MA 50 mg/kg; sham and crush controls received 1 mL DMSO vehicle.
“Rapamycin and 3-MA were obtained from Sigma-Aldrich... and dissolved in 0.5 % dimethyl sulfoxide (DMSO). Animals received daily intraperitoneal injections of rapamycin at a dose of 1 mg/kg... 3-MA at a dose of 50 mg/kg... or DMSO... for 5 days after the surgery.”
Dissect injured sciatic nerves at the defined post-injury time points, perfuse through the left ventricle with cold saline followed by 4% paraformaldehyde in PBS, then remove and freeze the injured nerve for histology or western blot analysis.
Note: Tissues were collected 1 and 6 weeks post-injury, n = 6 per group and time point.
“Sciatic nerves were dissected and harvested 1 and 6 weeks post-injury... Animals were decapitated and perfused via the left ventricle with cold saline followed by 4 % paraformaldehyde in 0.01 M phosphate-buffered saline (PBS; pH 7.35).”
Prepare 10-?m frozen transverse sections, wash in PBS, block in goat serum and BSA, incubate overnight with MBP, NF-200, or LC3B-II primary antibodies, follow with Alexa 488- or Cy3-conjugated secondary antibodies, and visualize sections on a Leica DM6000B fluorescence microscope at 400x magnification.
“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...)... then incubated with Alexa 488- or Cy3-conjugated secondary antibodies (1:1000; Invitrogen...)... visualized under an epifluorescence microscope (Leica, DM6000B...)”
Homogenize lesion-site nerve tissue in lysis buffer, quantify protein with a bicinchoninic assay kit, mix the lysate with sample buffer, separate 20?g per sample on 15% acrylamide gel, transfer by electroblotting, then detect LC3B-II and quantify the blots with Quantity One after imaging on Scanmaker 3836.
“A total of 20?g of each sample was separated on a 15 % acrylamide gel and transferred to a nitrocellulose membrane... by electroblotting (Bio-Rad...) at 120 V for 1.5 h followed by 70 V for 0.5 h at 4?C. The blots were visualized using Scanmaker 3836... and quantified with Quantity One software (Bio-Rad).”
Fix sciatic nerve segments in glutaraldehyde, post-fix in osmium tetroxide, dehydrate through graded alcohol and propylene oxide, embed in Epon, cut ultrathin sections on a Leica Ultracut R ultramicrotome, stain with uranyl acetate and lead citrate, and image on a JEOL JEM-1010 electron microscope.
“Sciatic nerve segments were removed 1 week after NCI and fixed with 2.5 % glutaraldehyde overnight at 4?C... post-fixed in 1 % osmium tetroxide for 1 h at 4?C... Ultrathin (70-nm) sections were prepared on an ultramicrotome (Ultracut R, Leica...)... visualized using an a JEM-1010 electron microscope (Jeol...)”
Use the Beyotime TUNEL apoptosis assay kit on 10-?m frozen sections from the lesion site, counterstain with DAPI, image at 400x magnification, and count TUNEL-positive cells in each section.
Note: Assay performed 1 week post-injury.
“TUNEL staining using the TUNEL apoptosis assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol 1 week post-injury. Samples were counterstained with DAPI... and imaged at 400? magnification.”
Blind the experimenters, then record stand time and footprint intensity at 1, 2, 3, and 6 weeks after injury using the CatWalk system with a GP-3360 high-speed video camera and 8.5-mm wide-angle lens under the illuminated walkway.
Note: Behavioral testing at 1, 2, 3, and 6 weeks after injury.
“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 camera (Gevicam...) equipped with an 8.5-mm wide-angle lens (Fujicon Corp...) positioned underneath the walkway automatically recorded paw prints.”
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.
Induce sciatic nerve crush injury in rats, pharmacologically modulate autophagy with rapamycin or 3-methyladenine, and quantify motor recovery and tissue-level regeneration across behavioral, immunocytochemical, western blot, electron microscopy, and TUNEL assays.
Objective
Induce sciatic nerve crush injury in rats, pharmacologically modulate autophagy with rapamycin or 3-methyladenine, and quantify motor recovery and tissue-level regeneration across behavioral, immunocytochemical, western blot, electron microscopy, and TUNEL assays.
Subjects
From paperrat • Sprague-Dawley • female • Adult • 180-220g
Cohort notes
From paperAnimals were purchased from the Laboratory Animal Centre at Southern Medical University and housed on a 12:12 h light/dark cycle with free access to food and water.
Induce sciatic nerve crush injury
Dose rapamycin, 3-MA, or vehicle after surgery (Daily for 5 days post-surgery)
Harvest and fix sciatic nerve tissue
Run immunocytochemistry on frozen nerve sections (Primary incubation 12 h at 4 C; secondary incubation 2 h at room temperature)
Stand time on CatWalk gait analysis
From paperComparisons within groups were made by one-way ANOVA, and between-group comparisons were made by one-way ANOVA followed by Bonferroni-corrected post hoc comparisons.
Artifact type
Longitudinal gait metrics and per-animal performance tables
Comparison focus
Compare recovery trajectory across post-injury timepoints and treatment conditions
Footprint intensity on CatWalk gait analysis
From paperComparisons within groups were made by one-way ANOVA, and between-group comparisons were made by one-way ANOVA followed by Bonferroni-corrected post hoc comparisons.
Artifact type
Longitudinal gait metrics and per-animal performance tables
Comparison focus
Compare recovery trajectory across post-injury timepoints and treatment conditions
LC3-II expression by western blot and immunocytochemistry
From paperComparisons within groups were made by one-way ANOVA, and between-group comparisons were made by one-way ANOVA followed by Bonferroni-corrected post hoc comparisons.
Artifact type
Longitudinal gait metrics and per-animal performance tables
Comparison focus
Compare recovery trajectory across post-injury timepoints and treatment conditions
MBP and NF-200 fluorescence intensity
From paperComparisons within groups were made by one-way ANOVA, and between-group comparisons were made by one-way ANOVA followed by Bonferroni-corrected post hoc comparisons.
Artifact type
Longitudinal gait metrics and per-animal performance tables
Comparison focus
Compare recovery trajectory across post-injury timepoints and treatment conditions
Stand time on CatWalk gait analysis
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
Footprint intensity on CatWalk gait analysis
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
LC3-II expression by western blot and immunocytochemistry
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
MBP and NF-200 fluorescence intensity
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 run-level gait data for each animal and preserve the timepoint or treatment labeling.
Preprocessing / cleaning
Comparisons within groups were made by one-way ANOVA, and between-group comparisons were made by one-way ANOVA followed by Bonferroni-corrected post hoc comparisons.
Scoring or quantification
Quantify the primary readouts for this experiment: Stand time on CatWalk gait analysis; Footprint intensity on CatWalk gait analysis; LC3-II expression by western blot and immunocytochemistry; MBP and NF-200 fluorescence intensity.
Normalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Stand time on CatWalk gait analysis, Footprint intensity on CatWalk gait analysis, LC3-II expression by western blot and immunocytochemistry, MBP and NF-200 fluorescence intensity.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Hai-cheng Huang et al. (2016). Autophagy Promotes Peripheral Nerve Regeneration and Motor Recovery Following Sciatic Nerve Crush Injury in Rats. Journal of Molecular Neuroscience
Induce sciatic nerve crush injury • Animals and Surgical Procedures
“rats were deeply anesthetized with sodium pentobarbital (50 mg/kg body weight by intraperitoneal injection), and the sciatic nerve in the right mid-thigh was exposed and clamped with a pair of forceps three times for 10 s each at 10-s intervals”
Dose rapamycin, 3-MA, or vehicle after surgery • Drug Treatment
“Rapamycin and 3-MA were obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 0.5 % dimethyl sulfoxide (DMSO).”
Dose rapamycin, 3-MA, or vehicle after surgery • Drug Treatment
“Animals received daily intraperitoneal injections of rapamycin at a dose of 1 mg/kg... 3-MA at a dose of 50 mg/kg... for 5 days after the surgery.”
Harvest and fix sciatic nerve tissue • Tissue Preparation
“Animals were decapitated and perfused via the left ventricle with cold saline followed by 4 % paraformaldehyde in 0.01 M phosphate-buffered saline (PBS; pH 7.35).”
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Assess motor recovery with CatWalk gait analysis: Blind the experimenters, then record stand time and footprint intensity at 1, 2, 3, and 6 weeks after injury using the CatWalk system with a GP-3360 high-speed video camera and...
Noldus
Original vendor
Verified vendor page
~$83,160.00
Regional market listing converted from KRW
Open vendor pageConductScience
ConductScience
Verified vendor page • reviewed Mar 10, 2026
$2,610.00
Price last checked Feb 17, 2026
SKU ME-5050
Open vendor pageWhy this match
Both systems support walkway-based gait analysis with automated paw-print capture and longitudinal motor recovery tracking in rodents.
Run immunocytochemistry on frozen nerve sections: Prepare 10-?m frozen transverse sections, wash in PBS, block in goat serum and BSA, incubate overnight with MBP, NF-200, or LC3B-II primary antibodies, follow with Alexa 488- or...
Why this match
The ConductScience fluorescence microscope covers the same immunofluorescence imaging use case, but it is a functional substitute rather than a Leica DM6000B configuration match.
Measure LC3-II by western blot: Homogenize lesion-site nerve tissue in lysis buffer, quantify protein with a bicinchoninic assay kit, mix the lysate with sample buffer, separate 20?g per sample on 15% acrylami...
ConductScience
ConductScience
Verified vendor page • reviewed Mar 10, 2026
$5,602.00
Price last checked Feb 17, 2026
SKU BB-BK-AG100
Open vendor pageWhy this is not exact
ReplicateScience links the mapped ConductScience imaging system for this western blot capture workflow, but a verified original product page has not been confirmed yet.
Run immunocytochemistry on frozen nerve sections: Prepare 10-?m frozen transverse sections, wash in PBS, block in goat serum and BSA, incubate overnight with MBP, NF-200, or LC3B-II primary antibodies, follow with Alexa 488- or...
Cell Signaling Technology
Original vendor page
Review specification
Cited manufacturer direct product page for LC3B antibody #3868.
Review vendor pageWhy this is not exact
The source paper cites Cell Signaling Technology for LC3B-II. This direct vendor page is included to speed reagent review, but the exact paper lot and ordering pack size still need lab confirmation.
Induce sciatic nerve crush injury: Deeply anesthetize adult female Sprague-Dawley rats with sodium pentobarbital, expose the right sciatic nerve in the mid-thigh, and create crush injury by clamping it three time...
Why this is not exact
This protocol needs the exact jaw geometry and crush workflow confirmed before linking any product page. ReplicateScience intentionally holds this item as review-needed until a verified instrument match is available.
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ConductScience • RWD-SP0003-M/RWD-SP0003-R
Leica • DM6000B
Bio-Rad
Bio-Rad
Microtek Technology Co. Ltd. • Scanmaker 3836
Link or price checked Feb 17, 2026
Sigma-Aldrich
Sigma-Aldrich
Sigma
Cell Signaling Technology
Invitrogen
Beyotime Institute of Biotechnology
Bio-Rad
GraphPad Software, Inc.
6 items with ReplicateScience direct pages
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