A robust neuromuscular system protects rat and human skeletal muscle from sarcopenia methods
Aim. Evidence-backed execution summary for A robust neuromuscular system protects rat and human skeletal muscle from sarcopenia methods from A robust neuromuscular system protects rat and human skeletal muscle from sarcopenia.
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Nerve micro-array analysis
reagent used in the protocol.
- Use
- Total RNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instruction. The quality of RNA samples was checked using the Standard Sensitivity RNA Analysis Kit on a Fragment Analyzer (Advanced Analytical Technologies). All cRNA targets were synthesized using the Illumina TotalPrep-96...
METHODS
reagent used in the protocol.
- Use
- Electromyography (EMG) measurements were performed on rats under isoflurane anesthesia. The left limbs were shaved and recording needle electrodes (twisted pairs, wire 150cm, needle 0.4 × 13mm, Neurolite, Switzerland) were sequentially placed into the gastrocnemius, tibialis anterior, triceps brachii and biceps...
Immunofluorescence staining and microscopy
reagent used in the protocol.
- Use
- For fiber type analysis, tibialis anterior and biceps brachii were frozen in isopentane cooled in liquid nitrogen, stored at -80°C and cryo-sectioned at 10µm. Sections were incubated 2h in blocking solution made with 4% IgG-free bovine serum albumin (001-000-162, Jackson immunoresearch) + 1% fetal bo...
Immunofluorescence staining and microscopy
reagent used in the protocol.
- Use
- For neuromuscular junction analyses, muscles were pinned on cork in PBS and injected with α-bungarotoxin-A488 (1:5000, B-13422 Thermo Fischer) for 30min to stain for acetylcholine receptors. Muscles were then rinsed with PBS and injected with 2% paraformaldehyde solution (P6148, Sigma Aldrich) for 15min, rinsed...
Motoneuron retrotracing study
reagent used in the protocol.
- Use
- Rats were anesthesized using isoflurane and the right legs were shaved and disinfected with ethanol. Fluorogold (Fluorochrome LLC) was diluted at 2% (w/v) and injected into the gastrocnemius and triceps brachii at 4 to 6 points of injection. The total volume of injection was 60-80 µL per muscle. Ten days later,...
Gene expression profiling
reagent used in the protocol.
- Use
- Total RNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instruction. RNA quantity was measured with Ribogreen (Life Technologies) and RNA quality was assessed on a Bioanalyzer (Agilent Technologies). Sequencing libraries were prepared from 250ng total RNA using the TruSeq RNA Lib...
Reverse transcription and qPCR
reagent used in the protocol.
- Use
- Total RNA was extracted using a commercial kit (miRNeasy Mini Kit, Qiagen) according to the manufacturer's instruction, and the concentration was measured using a NanoDrop device. Total RNA extracts were diluted at 20ng/ml and reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Appl...
Surface EMG
reagent used in the protocol.
- Use
- The maximal compound muscle action potential (CMAP) was evoked by a manually triggered stimulator (model DS7A; Digitimer, Welwyn Garden City, Hertfordshire, UK) using percutaneous stimulation (Medserve, Daventry, UK). For VL, stimulation was applied to the femoral nerve and a carbon-rubber anode electrode (Dermatrod...
Transcriptomic profiling reveals a neuromuscular signature specifically regulated in muscles susceptible to sarcopenia
Given the regional susceptibility to sarcopenia in rats (Figure ), differential profiling of hindlimb and forelimb muscle gene expression by RNA sequencing during aging provided a way to dissociate the pathways which are specifically regulated during sarcopenia only in hindlimb muscles, from those commonly regulated...
- Use
- Given the regional susceptibility to sarcopenia in rats (Figure ), differential profiling of hindlimb and forelimb muscle gene expression by RNA sequencing during aging provided a way to dissociate the pathways which are specifically regulated during sarcopenia only in hindlimb muscles, from those commonly regulated...
Neuromuscular defects with age are directly associated with the susceptibility to sarcopenia
The molecular and electrophysiological defects of regional neuromuscular decline between hindlimbs and forelimbs were then investigated at the cellular level. Increased fragmentation of the neuromuscular endplates through dispersion of acetylcholine receptors is believed to be a hallmark of impaired NMJ functionalit...
- Use
- The molecular and electrophysiological defects of regional neuromuscular decline between hindlimbs and forelimbs were then investigated at the cellular level. Increased fragmentation of the neuromuscular endplates through dispersion of acetylcholine receptors is believed to be a hallmark of impaired NMJ functionalit...
Nerve micro-array analysis
Total RNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instruction. The quality of RNA samples was checked using the Standard Sensitivity RNA Analysis Kit on a Fragment Analyzer (Advanced Analytical Technologies). All cRNA targets were synthesized using the Illumina TotalPrep-96...
- Use
- Total RNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instruction. The quality of RNA samples was checked using the Standard Sensitivity RNA Analysis Kit on a Fragment Analyzer (Advanced Analytical Technologies). All cRNA targets were synthesized using the Illumina TotalPrep-96...
METHODS
Electromyography (EMG) measurements were performed on rats under isoflurane anesthesia. The left limbs were shaved and recording needle electrodes (twisted pairs, wire 150cm, needle 0.4 × 13mm, Neurolite, Switzerland) were sequentially placed into the gastrocnemius, tibialis anterior, triceps brachii and biceps...
- Use
- Electromyography (EMG) measurements were performed on rats under isoflurane anesthesia. The left limbs were shaved and recording needle electrodes (twisted pairs, wire 150cm, needle 0.4 × 13mm, Neurolite, Switzerland) were sequentially placed into the gastrocnemius, tibialis anterior, triceps brachii and biceps...
METHODS
Gait parameters were measured using the Catwalk XT system (Noldus, Netherlands). Animals were placed to spontaneously walk on a glass lane with tangential illumination and each step was recorded by detecting light diffraction using a camera below the lane. At least 3 runs per animal were acquired with 10 minutes bre...
- Use
- Gait parameters were measured using the Catwalk XT system (Noldus, Netherlands). Animals were placed to spontaneously walk on a glass lane with tangential illumination and each step was recorded by detecting light diffraction using a camera below the lane. At least 3 runs per animal were acquired with 10 minutes bre...
Immunofluorescence staining and microscopy
For fiber type analysis, tibialis anterior and biceps brachii were frozen in isopentane cooled in liquid nitrogen, stored at -80°C and cryo-sectioned at 10µm. Sections were incubated 2h in blocking solution made with 4% IgG-free bovine serum albumin (001-000-162, Jackson immunoresearch) + 1% fetal bo...
- Use
- For fiber type analysis, tibialis anterior and biceps brachii were frozen in isopentane cooled in liquid nitrogen, stored at -80°C and cryo-sectioned at 10µm. Sections were incubated 2h in blocking solution made with 4% IgG-free bovine serum albumin (001-000-162, Jackson immunoresearch) + 1% fetal bo...
Immunofluorescence staining and microscopy
For neuromuscular junction analyses, muscles were pinned on cork in PBS and injected with α-bungarotoxin-A488 (1:5000, B-13422 Thermo Fischer) for 30min to stain for acetylcholine receptors. Muscles were then rinsed with PBS and injected with 2% paraformaldehyde solution (P6148, Sigma Aldrich) for 15min, rinsed...
- Use
- For neuromuscular junction analyses, muscles were pinned on cork in PBS and injected with α-bungarotoxin-A488 (1:5000, B-13422 Thermo Fischer) for 30min to stain for acetylcholine receptors. Muscles were then rinsed with PBS and injected with 2% paraformaldehyde solution (P6148, Sigma Aldrich) for 15min, rinsed...
Gene expression profiling
Total RNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instruction. RNA quantity was measured with Ribogreen (Life Technologies) and RNA quality was assessed on a Bioanalyzer (Agilent Technologies). Sequencing libraries were prepared from 250ng total RNA using the TruSeq RNA Lib...
- Use
- Total RNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instruction. RNA quantity was measured with Ribogreen (Life Technologies) and RNA quality was assessed on a Bioanalyzer (Agilent Technologies). Sequencing libraries were prepared from 250ng total RNA using the TruSeq RNA Lib...
Gene expression profiling
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Total RNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instruction. RNA quantity was measured with Ribogreen (Life Technologies) and RNA quality was assessed on a Bioanalyzer (Agilent Technologies). Sequencing libraries were prepared from 250ng total RNA using the TruSeq RNA Lib...
Statistics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All genome wide statistical analyses were performed using R version 3.1.3 and relevant Bioconductor packages as described in the gene expression profiling section. All other statistical analyses were performed using the GraphPad Prism software, and statistical significance was assessed by the Mann-Whitney test for b...
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Neuromuscular defects with age are directly associated with the susceptibility to sarcopenia
( A - B ) Neuromuscular fragmentation was measured on EDL ( A ) and biceps brachii ( B ) from adult, early sarcopenic or sarcopenic rats after staining for acetylcholine receptors (AchR) using fluorescently conjugat α-bungarotoxin. Representative images from each condition are shown. Scale bars represent 20µm. ( C ) Experimental design for retrotracer labeling studies. Motor innervation in gastrocnemius and triceps brachii was evaluated by injecting fluorogold into each muscle and quantifying its spinal cord accumulation 10 days later. ( D ) Number of motoneurons specifically innervating gastrocnemius and triceps brachii in adult and sarcopenic rats. For all graphs n=6-7 per group and an average of 50 NMJs were counted for each sample. * p<0.05 ** p<0.01 *** p<0.001 compared to the same category in adults, and # p<0.05 compared to the same category in early sarcopenic,...
METHODS
All experimental procedures on animals were in agreement with Swiss and EU ethical guidelines and approved by the local animal experimentation committee of the Canton of Vaud (license number VD2630). Male Wistar rats aged 8 months to 24 months were obtained from Janvier Labs (Le Genest-Saint-Isle, France). Upon arrival all animals were housed by two in standard type 4 cages with ad libitum access to food and water on a 12 hour light/dark cycle at a temperature between 20-24°C and a relative humidity between 50-60%. All rats labeled by age were grouped by date of birth within one month, and further grouping was then based on the muscle phenotype in hindlimbs under the following categories: adult (8-10 months of age), early-sarcopenic (18-20 months of age), and sarcopenic (22-24 months of age). Rats were sacrificed by exsanguination under isoflurane anesthesia and skeletal muscles...
METHODS
Electromyography (EMG) measurements were performed on rats under isoflurane anesthesia. The left limbs were shaved and recording needle electrodes (twisted pairs, wire 150cm, needle 0.4 × 13mm, Neurolite, Switzerland) were sequentially placed into the gastrocnemius, tibialis anterior, triceps brachii and biceps brachii muscles. Supra-maximal electric stimulation was achieved via stimulating needle electrodes sequentially placed around the sciatic nerve and the radial nerve, and the resulting compound muscle action potential (CMAP) was recorded using the Keypoint software (Neurolite, Switzerland).
METHODS
Gait parameters were measured using the Catwalk XT system (Noldus, Netherlands). Animals were placed to spontaneously walk on a glass lane with tangential illumination and each step was recorded by detecting light diffraction using a camera below the lane. At least 3 runs per animal were acquired with 10 minutes break in between. Locomotion patterns and gait speed were then analyzed from smooth runs, which were defined as runs during which the rat maintained constant speed for at least 3-4 prints per paw, using the automated Catwalk analysis software. All runs were required, manually edited for correct detection of the paws.
Immunofluorescence staining and microscopy
For neuromuscular junction analyses, muscles were pinned on cork in PBS and injected with α-bungarotoxin-A488 (1:5000, B-13422 Thermo Fischer) for 30min to stain for acetylcholine receptors. Muscles were then rinsed with PBS and injected with 2% paraformaldehyde solution (P6148, Sigma Aldrich) for 15min, rinsed again and stored at 4°C. Muscles were then separated into bundles of 20-30 fibers, mounted with fluorescence mounting medium (S302380, Dako) and pressed overnight at 4°C. Images were acquired on a Leica DMI 6000B microscope. For each muscle, approximately 50 neuromuscular junctions were imaged and each junction was classified into 3 classes (1-2 fragments, 3-4 fragments or ≥5 fragments) according to the number of fragments counted manually.
Motoneuron retrotracing study
Rats were anesthesized using isoflurane and the right legs were shaved and disinfected with ethanol. Fluorogold (Fluorochrome LLC) was diluted at 2% (w/v) and injected into the gastrocnemius and triceps brachii at 4 to 6 points of injection. The total volume of injection was 60-80 µL per muscle. Ten days later, animals were sacrificed by intracardial perfusion of paraformaldehyde and the spinal cord was dissected out, placed in paraformaldehyde solution overnight at 4°C and subsequently placed in 20% sucrose solution for 3-4 days. The cervical and lumbar regions of each spinal cord were then removed and frozen in OCT embedding medium (361603E, VWR Chemicals). Each sample was cryo-sectioned at 40µm on its entire length and 1 out of 4 sections were examined to count the number of labeled motoneurons.
Human clinical study
Muscle anatomical cross sectional area. Magnetic resonance imaging (MRI) was used to measure peak VL and TA muscle anatomical cross sectional areas using a T1-weighted turbo 3D sequence on a 0.25-T G-Scan (Esaote, Genoe, Italy). The motor point over the VL muscle belly was marked, and contiguous transverse-plane slices of 6mm thickness were collected. Images were analyzed off-line using Osirix imaging software (OsiriX medical imaging, OsiriX, Atlanta, USA) and the cross-sectional at the motor point was recorded.
Surface EMG
The maximal compound muscle action potential (CMAP) was evoked by a manually triggered stimulator (model DS7A; Digitimer, Welwyn Garden City, Hertfordshire, UK) using percutaneous stimulation (Medserve, Daventry, UK). For VL, stimulation was applied to the femoral nerve and a carbon-rubber anode electrode (Dermatrode self-adhering electrode, 5.08cm diameter, Farmadomo, NL) was placed over the skin overlying the gluteus muscle. For TA, the anode was placed on the medial knee joint cleft and stimulation was applied to a superficial part of the common peroneal nerve on the lateral aspect of the shank close to the head of the fibula. For each muscle, the recording surface-EMG electrode was positioned over the motor point of the respective muscle belly to ensure the largest CMAP (negative peak amplitude) with fastest rise-time. For both VL and TA, the stimulator voltage was fixed at 400V a...
Measurement outputs
What raw and processed outputs should exist?
Given the regional susceptibility to sarcopenia in rats (Figure ), differential profiling of hindlimb and forelimb muscle gene expression by RNA sequencing during aging provided...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
RNA-Seq profiling was performed comparing gastrocnemius and triceps brachii from adult, early sarcopenic or sarcopenic rats. ( A ) Venn diagrams of genes significantly regulated...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Aging promotes a global up-regulation of many genes controlling NMJ assembly and function in hindlimb muscles, likely through de-repression of extra-synaptic gene inhibition [ ]...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
To further investigate whether intrinsic molecular defects in nerves could contribute to the susceptibility to sarcopenia, we performed a gene expression analysis comparing hind...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
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
( A, C ) Graphs showing the correlation between muscle cross-sectional area (CSA) and CMAP negative peak amplitude in vastus lateralis ( A ) and tibialis anterior ( C ) from adult (i.e 20-35 years) and elderly (i.e >65 years) men.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Given the regional susceptibility to sarcopenia in rats (Figure ), differential profiling of hindlimb and forelimb muscle gene expression by RNA sequencing during aging provided...; RNA-Seq profiling was performed comparing gastrocnemius and triceps brachii from adult, early sarcopenic or sarcopenic rats. ( A ) Venn diagrams of genes significantly regulated...; Aging promotes a global up-regulation of many genes controlling NMJ assembly and function in hindlimb muscles, likely through de-repression of extra-synaptic gene inhibition [ ]...; To further investigate whether intrinsic molecular defects in nerves could contribute to the susceptibility to sarcopenia, we performed a gene expression analysis comparing hind....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
( A, C ) Graphs showing the correlation between muscle cross-sectional area (CSA) and CMAP negative peak amplitude in vastus lateralis ( A ) and tibialis anterior ( C ) from ad...; RNA-Seq profiling was performed comparing gastrocnemius and triceps brachii from adult, early sarcopenic or sarcopenic rats. ( A ) Venn diagrams of genes significantly regulated...; ( A - B ) Neuromuscular fragmentation was measured on EDL ( A ) and biceps brachii ( B ) from adult, early sarcopenic or sarcopenic rats after staining for acetylcholine recepto...; ( A ) Venn diagram comparing the overlap of gene regulation with age between sciatic and radial nerves using a linear model. Genes were considered significantly regulated when t...
from paperReporting output
Report representative outputs alongside summary comparisons for Given the regional susceptibility to sarcopenia in rats (Figure ), differential profiling of hindlimb and forelimb muscle gene expression by RNA sequencing during aging provided..., RNA-Seq profiling was performed comparing gastrocnemius and triceps brachii from adult, early sarcopenic or sarcopenic rats. ( A ) Venn diagrams of genes significantly regulated..., Aging promotes a global up-regulation of many genes controlling NMJ assembly and function in hindlimb muscles, likely through de-repression of extra-synaptic gene inhibition [ ]..., To further investigate whether intrinsic molecular defects in nerves could contribute to the susceptibility to sarcopenia, we performed a gene expression analysis comparing hind....
inferred from protocolStructured statistical methods
( A, C ) Graphs showing the correlation between muscle cross-sectional area (CSA) and CMAP negative peak amplitude in vastus lateralis ( A ) and tibialis anterior ( C ) from ad...; RNA-Seq profiling was performed comparing gastrocnemius and triceps brachii from adult, early sarcopenic or sarcopenic rats. ( A ) Venn diagrams of genes significantly regulated...; ( A - B ) Neuromuscular fragmentation was measured on EDL ( A ) and biceps brachii ( B ) from adult, early sarcopenic or sarcopenic rats after staining for acetylcholine recepto...; ( A ) Venn diagram comparing the overlap of gene regulation with age between sciatic and radial nerves using a linear model. Genes were considered significantly regulated when t...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
( A - B ) Neuromuscular fragmentation was measured on EDL ( A ) and biceps brachii ( B ) from adult, early sarcopenic or sarcopenic rats after staining for acetylcholine receptors (AchR) using fluorescently conjugat α-bungarotoxin. Representative images from each condition are shown. Scale bars represent 20µm. ( C ) Experimental design for retrotracer labeling studies. Motor innervation in gastrocnemius and triceps brachii was evaluated by injecting fluorogold into each muscle and quantifying its spinal cord accumulation 10 days later. ( D ) Number of motoneurons specifically innervating gastrocnemius and triceps brachii in adult and sarcopenic rats. For all graphs n=6-7 per group and an average of 50 NMJs were counted for each sample. * p<0.05 ** p<0.01 *** p<0.001 compared to the same category in adults, and # p<0.05 compared to the same category in early sarcopenic, using a one-way ANOVA test.
All experimental procedures on animals were in agreement with Swiss and EU ethical guidelines and approved by the local animal experimentation committee of the Canton of Vaud (license number VD2630). Male Wistar rats aged 8 months to 24 months were obtained from Janvier Labs (Le Genest-Saint-Isle, France). Upon arrival all animals were housed by two in standard type 4 cages with ad libitum access to food and water on a 12 hour light/dark cycle at a temperature between 20-24°C and a relative humidity between 50-60%. All rats labeled by age were grouped by date of birth within one month, and further grouping was then based on the muscle phenotype in hindlimbs under the following categories: adult (8-10 months of age), early-sarcopenic (18-20 months of age), and sarcopenic (22-24 months of age). Rats were sacrificed by exsanguination under isoflurane anesthesia and skeletal muscles were dissected free of fat, weighed and snap frozen in liquid nitrogen or processed for histology as described below. A piece of about 1cm of sciatic and radial nerves was dissected out from the mid-thigh or arm region and snap frozen in liquid nitrogen.
Electromyography (EMG) measurements were performed on rats under isoflurane anesthesia. The left limbs were shaved and recording needle electrodes (twisted pairs, wire 150cm, needle 0.4 × 13mm, Neurolite, Switzerland) were sequentially placed into the gastrocnemius, tibialis anterior, triceps brachii and biceps brachii muscles. Supra-maximal electric stimulation was achieved via stimulating needle electrodes sequentially placed around the sciatic nerve and the radial nerve, and the resulting compound muscle action potential (CMAP) was recorded using the Keypoint software (Neurolite, Switzerland).
Gait parameters were measured using the Catwalk XT system (Noldus, Netherlands). Animals were placed to spontaneously walk on a glass lane with tangential illumination and each step was recorded by detecting light diffraction using a camera below the lane. At least 3 runs per animal were acquired with 10 minutes break in between. Locomotion patterns and gait speed were then analyzed from smooth runs, which were defined as runs during which the rat maintained constant speed for at least 3-4 prints per paw, using the automated Catwalk analysis software. All runs were required, manually edited for correct detection of the paws.
For neuromuscular junction analyses, muscles were pinned on cork in PBS and injected with α-bungarotoxin-A488 (1:5000, B-13422 Thermo Fischer) for 30min to stain for acetylcholine receptors. Muscles were then rinsed with PBS and injected with 2% paraformaldehyde solution (P6148, Sigma Aldrich) for 15min, rinsed again and stored at 4°C. Muscles were then separated into bundles of 20-30 fibers, mounted with fluorescence mounting medium (S302380, Dako) and pressed overnight at 4°C. Images were acquired on a Leica DMI 6000B microscope. For each muscle, approximately 50 neuromuscular junctions were imaged and each junction was classified into 3 classes (1-2 fragments, 3-4 fragments or ≥5 fragments) according to the number of fragments counted manually.
Rats were anesthesized using isoflurane and the right legs were shaved and disinfected with ethanol. Fluorogold (Fluorochrome LLC) was diluted at 2% (w/v) and injected into the gastrocnemius and triceps brachii at 4 to 6 points of injection. The total volume of injection was 60-80 µL per muscle. Ten days later, animals were sacrificed by intracardial perfusion of paraformaldehyde and the spinal cord was dissected out, placed in paraformaldehyde solution overnight at 4°C and subsequently placed in 20% sucrose solution for 3-4 days. The cervical and lumbar regions of each spinal cord were then removed and frozen in OCT embedding medium (361603E, VWR Chemicals). Each sample was cryo-sectioned at 40µm on its entire length and 1 out of 4 sections were examined to count the number of labeled motoneurons.
Muscle anatomical cross sectional area. Magnetic resonance imaging (MRI) was used to measure peak VL and TA muscle anatomical cross sectional areas using a T1-weighted turbo 3D sequence on a 0.25-T G-Scan (Esaote, Genoe, Italy). The motor point over the VL muscle belly was marked, and contiguous transverse-plane slices of 6mm thickness were collected. Images were analyzed off-line using Osirix imaging software (OsiriX medical imaging, OsiriX, Atlanta, USA) and the cross-sectional at the motor point was recorded.
The maximal compound muscle action potential (CMAP) was evoked by a manually triggered stimulator (model DS7A; Digitimer, Welwyn Garden City, Hertfordshire, UK) using percutaneous stimulation (Medserve, Daventry, UK). For VL, stimulation was applied to the femoral nerve and a carbon-rubber anode electrode (Dermatrode self-adhering electrode, 5.08cm diameter, Farmadomo, NL) was placed over the skin overlying the gluteus muscle. For TA, the anode was placed on the medial knee joint cleft and stimulation was applied to a superficial part of the common peroneal nerve on the lateral aspect of the shank close to the head of the fibula. For each muscle, the recording surface-EMG electrode was positioned over the motor point of the respective muscle belly to ensure the largest CMAP (negative peak amplitude) with fastest rise-time. For both VL and TA, the stimulator voltage was fixed at 400V and the pulse width at 50µS, and the current was then increased incrementally until the CMAP negative peak amplitude plateaued. At this point the current was increased again by 30mA to ensure supra-maximal stimulation (usually occurring between 100 and 200mA for VL and 80-120mA for TA).
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "A robust neuromuscular system protects rat and human skeletal muscle from sarcopenia methods",
"description": "Evidence-backed execution summary for A robust neuromuscular system protects rat and human skeletal muscle from sarcopenia methods from A robust neuromuscular system protects rat and human skeletal muscle from sarcopenia.",
"totalTime": "PT7480M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Neuromuscular defects with age are directly associated with the susceptibility to sarcopenia",
"text": "( A - B ) Neuromuscular fragmentation was measured on EDL ( A ) and biceps brachii ( B ) from adult, early sarcopenic or sarcopenic rats after staining for acetylcholine receptors (AchR) using fluorescently conjugat α-bungarotoxin. Representative images from each condition are shown. Scale bars represent 20µm. ( C ) Experimental design for retrotracer labeling studies. Motor innervation in gastrocnemius and triceps brachii was evaluated by injecting fluorogold into each muscle and quantifying its spinal cord accumulation 10 days later. ( D ) Number of motoneurons specifically innervating gastrocnemius and triceps brachii in adult and sarcopenic rats. For all graphs n=6-7 per group and an average of 50 NMJs were counted for each sample. * p<0.05 ** p<0.01 *** p<0.001 compared to the same category in adults, and # p<0.05 compared to the same category in early sarcopenic,..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "METHODS",
"text": "All experimental procedures on animals were in agreement with Swiss and EU ethical guidelines and approved by the local animal experimentation committee of the Canton of Vaud (license number VD2630). Male Wistar rats aged 8 months to 24 months were obtained from Janvier Labs (Le Genest-Saint-Isle, France). Upon arrival all animals were housed by two in standard type 4 cages with ad libitum access to food and water on a 12 hour light/dark cycle at a temperature between 20-24°C and a relative humidity between 50-60%. All rats labeled by age were grouped by date of birth within one month, and further grouping was then based on the muscle phenotype in hindlimbs under the following categories: adult (8-10 months of age), early-sarcopenic (18-20 months of age), and sarcopenic (22-24 months of age). Rats were sacrificed by exsanguination under isoflurane anesthesia and skeletal muscles..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "METHODS",
"text": "Electromyography (EMG) measurements were performed on rats under isoflurane anesthesia. The left limbs were shaved and recording needle electrodes (twisted pairs, wire 150cm, needle 0.4 × 13mm, Neurolite, Switzerland) were sequentially placed into the gastrocnemius, tibialis anterior, triceps brachii and biceps brachii muscles. Supra-maximal electric stimulation was achieved via stimulating needle electrodes sequentially placed around the sciatic nerve and the radial nerve, and the resulting compound muscle action potential (CMAP) was recorded using the Keypoint software (Neurolite, Switzerland)."
},
{
"@type": "HowToStep",
"position": 4,
"name": "METHODS",
"text": "Gait parameters were measured using the Catwalk XT system (Noldus, Netherlands). Animals were placed to spontaneously walk on a glass lane with tangential illumination and each step was recorded by detecting light diffraction using a camera below the lane. At least 3 runs per animal were acquired with 10 minutes break in between. Locomotion patterns and gait speed were then analyzed from smooth runs, which were defined as runs during which the rat maintained constant speed for at least 3-4 prints per paw, using the automated Catwalk analysis software. All runs were required, manually edited for correct detection of the paws."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Immunofluorescence staining and microscopy",
"text": "For neuromuscular junction analyses, muscles were pinned on cork in PBS and injected with α-bungarotoxin-A488 (1:5000, B-13422 Thermo Fischer) for 30min to stain for acetylcholine receptors. Muscles were then rinsed with PBS and injected with 2% paraformaldehyde solution (P6148, Sigma Aldrich) for 15min, rinsed again and stored at 4°C. Muscles were then separated into bundles of 20-30 fibers, mounted with fluorescence mounting medium (S302380, Dako) and pressed overnight at 4°C. Images were acquired on a Leica DMI 6000B microscope. For each muscle, approximately 50 neuromuscular junctions were imaged and each junction was classified into 3 classes (1-2 fragments, 3-4 fragments or ≥5 fragments) according to the number of fragments counted manually."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Motoneuron retrotracing study",
"text": "Rats were anesthesized using isoflurane and the right legs were shaved and disinfected with ethanol. Fluorogold (Fluorochrome LLC) was diluted at 2% (w/v) and injected into the gastrocnemius and triceps brachii at 4 to 6 points of injection. The total volume of injection was 60-80 µL per muscle. Ten days later, animals were sacrificed by intracardial perfusion of paraformaldehyde and the spinal cord was dissected out, placed in paraformaldehyde solution overnight at 4°C and subsequently placed in 20% sucrose solution for 3-4 days. The cervical and lumbar regions of each spinal cord were then removed and frozen in OCT embedding medium (361603E, VWR Chemicals). Each sample was cryo-sectioned at 40µm on its entire length and 1 out of 4 sections were examined to count the number of labeled motoneurons."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Human clinical study",
"text": "Muscle anatomical cross sectional area. Magnetic resonance imaging (MRI) was used to measure peak VL and TA muscle anatomical cross sectional areas using a T1-weighted turbo 3D sequence on a 0.25-T G-Scan (Esaote, Genoe, Italy). The motor point over the VL muscle belly was marked, and contiguous transverse-plane slices of 6mm thickness were collected. Images were analyzed off-line using Osirix imaging software (OsiriX medical imaging, OsiriX, Atlanta, USA) and the cross-sectional at the motor point was recorded."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Surface EMG",
"text": "The maximal compound muscle action potential (CMAP) was evoked by a manually triggered stimulator (model DS7A; Digitimer, Welwyn Garden City, Hertfordshire, UK) using percutaneous stimulation (Medserve, Daventry, UK). For VL, stimulation was applied to the femoral nerve and a carbon-rubber anode electrode (Dermatrode self-adhering electrode, 5.08cm diameter, Farmadomo, NL) was placed over the skin overlying the gluteus muscle. For TA, the anode was placed on the medial knee joint cleft and stimulation was applied to a superficial part of the common peroneal nerve on the lateral aspect of the shank close to the head of the fibula. For each muscle, the recording surface-EMG electrode was positioned over the motor point of the respective muscle belly to ensure the largest CMAP (negative peak amplitude) with fastest rise-time. For both VL and TA, the stimulator voltage was fixed at 400V a..."
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"name": "Transcriptomic profiling reveals a neuromuscular signature specifically regulated in muscles susceptible to sarcopenia"
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"name": "Neuromuscular defects with age are directly associated with the susceptibility to sarcopenia"
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