Source Paper
Alexandre Henriques, Vincent Croixmarie, David A. Priestman, Angela Rosenbohm, Sylvie Dirrig-Grosch et al.
Human Molecular Genetics • 2015
Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset disease characterized by upper and lower motor neuron degeneration, muscle wasting and paralysis. Growing evidence suggests a link between changes in lipid metabolism and ALS. Here, we used UPLC/TOF-MS to survey the lipidome in SOD1(G86R) mice, a model of ALS. Significant changes in lipid expression were evident in spinal cord and skeletal muscle before overt neuropathology. In silico analysis also revealed appreciable changes in sphingolipids including ceramides and glucosylceramides (GlcCer). HPLC analysis showed increased amounts of GlcCer and downstream glycosphingolipids (GSLs) in SOD1(G86R) muscle compared with wild-type littermates. Glucosylceramide synthase (GCS), the enzyme responsible for GlcCer biosynthesis, was up-regulated in muscle of SOD1(G86R) mice and ALS patients, and in muscle of wild-type mice after surgically induced denervation. Conversely, inhibition of GCS in wild-type mice, following transient peripheral nerve injury, reversed the overexpression of genes in muscle involved in oxidative metabolism and delayed motor recovery. GCS inhibition in SOD1(G86R) mice also affected the expression of metabolic genes and induced a loss of muscle strength and morphological deterioration of the motor endplates. These findings suggest that GSLs may play a critical role in ALS muscle pathology and could lead to the identification of new therapeutic targets.
Objective: To induce sciatic nerve crush injury in mice and study motor recovery and nerve regeneration, with comparison between pre-symptomatic and symptomatic disease states
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FVB/N male mice overexpressing SOD1(G86R) maintained in animal facility with controlled environment
Note: 12 h light/dark cycle; water and A04 rodent chow provided ad libitum
“FVB/N male mice, overexpressing SOD1(G86R), were maintained in our animal facility at 23°C with a 12 h light/dark cycle. Mice had water and regular A04 rodent chow ad libitum”
Mice fasted overnight before sacrifice
“were fasted overnight before sacrifice”
Anesthetize mice with ketamine chlorohydrate and xylazine
Note: Dosages: ketamine 100 mg/kg, xylazine 5 mg/kg
“mice were anesthetized with ketamine chlorohydrate (100 mg/kg) and xylazine (5 mg/kg)”
Expose the sciatic nerve at mid-thigh level through surgical incision
Note: Performed at mid-thigh level
“The sciatic nerve was exposed at mid-thigh level”
Crush the exposed sciatic nerve using fine forceps
Note: Alternative procedure: 3-mm section can be removed with microscissors instead of crushing
“crushed with fine forceps for 30 s, or a 3-mm section removed with microscissors”
Close the skin incision with sutures
“The skin incision was sutured, and mice were allowed to recover”
Allow mice to recover from anesthesia and surgery
Note: Contralateral hind limb serves as control
“mice were allowed to recover. The hind limb, contralateral to the lesion, served as control”
Administer AMP-DNM via daily intraperitoneal injections to inhibit GCS enzymatic activity
Note: Dose: 25 mg/kg daily in 0.9% NaCl containing 5% DMSO; 10 days is typical timeframe for motor function recovery
“mice subjected to sciatic nerve crush were treated with daily intraperitoneal injections of N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM, Cayman Chemical Company, Ann Arbor, MI) for 10 days, the typical time frame required for recovery of motor function under normal conditions”
Euthanize mice by decapitation after deep anesthesia
Note: Sodium pentobarbital dose: 120 mg/kg
“Mice were killed by decapitation after deep anesthesia with 120 mg/kg sodium pentobarbital”
Rapidly dissect lumbar spinal cord and muscle tissue; freeze in liquid nitrogen and store at -80°C
Note: For immunolabeling experiments, muscle samples are fixed with 4% paraformaldehyde and stored in PBS at 4°C instead
“Lumbar spinal cord and muscle were rapidly dissected, frozen in liquid nitrogen and stored at -80°C. For immunolabeling experiments, dissected muscle samples were fixed with 4% paraformaldehyde and stored in PBS at 4°C”
FVB/N male mice overexpressing SOD1(G86R); WT male littermates served as controls
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