Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

Summary

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

Abstract

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

Introduction

Many peripheral nerve injury models exist, but one that stands out for the study of motoneurons is the facial nerve axotomy model. The facial nerve, also known as cranial nerve VII, originates in the pons and innervates the muscles of facial expression^1,2. In this surgical protocol, the facial nerve is exposed at its exit from the stylomastoid foramen and either cut or crushed. The severity of nerve injury can be classified following the Sunderland³ classifications, which differentiates the injury based on the intactness of the axons, endoneurium, perineurium, and epineurium, which are connective tissue layers that sequentially wrap around the ax....

Protocol

All procedures executed are approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee and follow National Institute of Health guidelines.

1. Surgical Technique

1. Maintain aseptic technique during this procedure by using sterile gloves, instruments, and a sterile surgical field according to NIH guidelines²⁵. Sterilize tools before beginning surgery by autoclaving them (see Table of Specific Reagents/Equipment for complete list). Use a glass bead sterilizer to sterilize tools during the operation.

2. Anesthesia and Preparation

Results

After the facial nerve axotomy is performed, motoneuron loss occurs as a result of the injury. Motoneuron survival after injury depends on many variables, such as gender, animal age at time of surgery, and the timepoint at which the motoneuron counts are done, and the Moran and Graeber review²⁴ and Jinno and Yamada review²² both summarize motoneuron survival data. Typically, about 86% of motoneurons survive at 28 days post-axotomy^14,15,26. Kinetics of motoneuron loss are described in Serp.......

Discussion

The critical step for this protocol is positioning the mouse properly before surgery is begun. If the mouse is not lying flat on its side, the ear is not taped at the correct angle, or the incision is made in an incorrect location, then finding the facial nerve becomes much more difficult. When this technique is mastered, surgeries will take only minutes per mouse.

Either sutures, glue, or wound clips can be used to close the wound. Wound clips are preferred because of the small size of incis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereo microscope	Leica	M60
Labeling tape	Fisher Scientific	15-952
Vannas-Tübingen spring scissors - straight/sharp/8.5 cm/5 mm cutting edge	Fine Science Tools	15003-08	Sterilize before use
Dumont #5/45 forceps - standard tips/angled 45°/Dumoxel/11 cm	Fine Science Tools	11251-35	Sterilize before use
Michel suture clips - 7.5 mm x 1.75 mm	Fine Science Tools	12040-01	Described as "wound clip" in protocol, sterilize before use
Hagenbarth cross action wound clip applier 5"	George Tiemann & Co	160-910	Used to apply wound clip, sterilize before use
Michel suture clip applicator & remover - For 7.5 mm clips	Fine Science Tools	12029-12	Used to remove wound clip
0.9% Sodium chloride injection, USP	Hospira	0409-4888-10
Betadine, 16 oz, with dispenser	Fisher Scientific	19-027132
70% Ethanol
Glass bead sterilizer