A robust neuromuscular system protects rat and human skeletal muscle from sarcopenia methods
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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
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Gait recovery and locomotor function
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- 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
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- Group comparisons of gait indices, stride metrics, or recovery curves
Microscopy-based tissue or cell-state imaging
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- Field or section images captured from matched samples
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- Selected representative panels with quantified intensity, counts, or area measurements
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- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
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Acquisition
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Quantify the primary readouts for this experiment: Gait recovery and locomotor function; Microscopy-based tissue or cell-state imaging.
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inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function, Microscopy-based tissue or cell-state imaging.
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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
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