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Abstract

Introduction

Protocol

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Acknowledgements

Materials

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Neuroscience

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

Published: August 11th, 2016

DOI:

10.3791/54186

1Carilion Research Institute, Virginia Tech, 2Carilion School of Medicine, Virginia Tech, 3Department of Biological Sciences, Virginia Tech

We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle.

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative to understand the cellular and molecular changes involved in maintaining and repairing NMJs. For this purpose, we have developed a method to reliably and consistently examine regenerating NMJs in mice. This nerve injury method involves crushing the common fibular nerve as it passes over the lateral head of the gastrocnemius muscle tendon near the knee. Using 70 day old female mice, we demonstrate that motor axons begin to reinnervate previous postsynaptic targets within 7 days post-crush. They completely reoccupy their previous synaptic areas by 12 days. To determine the reliability of this injury method, we compared reinnervation rates between individual 70 day old female mice. We found that the number of reinnervated postsynaptic sites was similar between mice at 7, 9, and 12 days post-crush. To determine if this injury assay can also be used to compare molecular changes in muscles, we examined levels of the gamma-subunit of the muscle nicotinic receptor (gamma-AChR) and the muscle-specific kinase (MuSK). The gamma-AChR subunit and MuSK to are highly upregulated following denervation and return to normal levels following reinnervation of NMJs. We found a close relationship between transcript levels for these genes and innervation status of muscles. We believe that this method will accelerate our understanding of the cellular and molecular changes involved in repairing the NMJ and other synapses.

In young adult and healthy animals, the neuromuscular junction (NMJ) is a highly stable connection between the presynapse, the nerve ending of an α-motor axon, and the postsynapse, the specialized region of an extrafusal muscle fiber where nicotinic acetylcholine receptors (AChRs) selectively aggregate1. The nearly perfect apposition of the pre- and post-synaptic apparatuses is necessary for proper neurotransmission, survival of α-motor neurons and muscle fibers and motor function. Unfortunately, the function of the NMJ is adversely affected by aging, diseases such as amyotrophic lateral sclerosis (ALS), autoimmune diseases and injury to muscles....

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All experiments were carried out under NIH guidelines and animal protocols approved by the Virginia Tech Institutional Animal Care and Use Committee.

1. Preparing Animals for Surgery

  1. Anesthetize mice with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) via subcutaneous inguinal injection with a sterile 1 ml insulin syringe. Carrier solution contains a mixture of 0.9% saline, 17.4 mg/ml ketamine, and 2.6 mg/ml xylazine. Place animals back in cages while waiting for medicati.......

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The common fibular nerve, also called the common peroneal nerve, arises from the sciatic nerve above the popliteal fossa, where it swings around the head of the fibula to the anterior aspect of the leg (Figure 1A). There it branches into the superficial and deep fibular nerves, together supplying the dorsiflexors of the foot and toes (anterior tibialis, extensor digitorum longus and brevis, and extensor halluces longus muscles), and the everters of the foot (peroneus musc.......

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The method presented in this manuscript provides unique opportunities to identify mechanisms involved in repairing neuromuscular junctions (NMJ). This method involves crushing the common fibular nerve as it passes over the gastrocnemius tendon near the knee. We show that after only 5 sec of nerve compression with a forceps, complete degeneration is noted by 4 days after injury. In young adult mice, alpha-motor axons begin to reinnervate previous synaptic sites in the extensor digitorum longus muscle (EDL) at 7 days post-.......

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The authors thank members of the Valdez laboratory for intellectual input on experiments and comments on the manuscript.

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Name Company Catalog Number Comments
Ketamine VetOne  501072 
Xylazine Lloyd Inc.  003437 
Buprenorphine  Zoopharm 1Z-73000-150910 
Nair Nair
Kim-wipes Kimtech 34155
Electric Razor Braintree Scientific CLP-64800
80% EtOH/H20
10% Proviodine
1 mL Insulin Syringe
Spring Scissors Vannas 91500-09
No. 15 scalpel Braintree Scientific SSS 15
#5 Forceps Dumont 11252-00
6-0 silk suture on reverse cutting needle  Suture Express 752B 
Rodent Heating Pad Braintree Scientific AP-R-18.5
Alexa 555 conjugated alpha-BTX Molecular Probes B35451
Vectashield Vector Labs H-1000
Olympus Stereo Zoom Microscope Olympus 562037192
Zeiss 700 Confocal Microscope Zeiss
Variable-flow peristaltic perfusion pump Fisher Scientific 13-876-3
Aurum Total RNA Mini Kit Bio-Rad 7326820
Bio-Rad iScript RT Supermix Bio-Rad 1708840
SsoFast Evagreen Supermix Bio-Rad 1725200
Bio-Rad CFX96 Bio-Rad 1855196
Puralube vet ointment Puralube 1621
Synaptotagmin-2 antibody Antibodies-Online ABIN401605
Neurofilament antibody Antibodies-Online ABIN2475842

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