Name: facial nerve axotomy in mice: a model to study motoneuron response to injury
Uploaded: 2015-02-23T15:00:00
Description: Watch this Scientific Journal Video about Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury at JoVE.com

This work is funded by NIH RO1 NS 40433 (K.J.J.).

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
<ol>
	<li>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 comp...

<ol>
	<li>Kaufman, M., Bard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Anatomical Basis of Mouse Development. , (1999).</li><li>Ashwell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+mouse+facial+nerve+nucleus:+morphology+and+musculotopic+organization.">The adult mouse facial nerve nucleus: morphology and musculotopic organization.</a> Journal of Anatomy. 135, 531-538 (1982).</li><li>Sunderland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+classification+of+peripheral+nerve+injuries+producing+loss+of+function.">A classification of peripheral nerve injuries producing loss of function.</a> Brain : A Journal Of Neurology. 74, 491-516 (1951).</li><li>Beahrs, T., Tanzer, L., Sanders, V. M., Jones, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recovery+and+facial+motoneuron+survival+are+influenced+by+immunodeficiency+in+crush-axotomized+mice.">Functional recovery and facial motoneuron survival are influenced by immunodeficiency in crush-axotomized mice.</a> Experimental Neurology. 221, 225-230 (2010).</li><li>Mesnard, N. A., Haulcomb, M. M., Tanzer, L., Sanders, V., Jones, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+functional+recovery+in+presymptomatic+mSOD1G93A+mice+following+facial+nerve+crush+axotomy.">Delayed functional recovery in presymptomatic mSOD1G93A mice following facial nerve crush axotomy.</a> Journal of Neurodegeneration &amp; Regeneration. 4, 21-25 (2013).</li><li>Komiyama, M., Shibata, H., Suzuki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatotopic+representation+of+facial+muscles+within+the+facial+nucleus+of+the+mouse.+A+study+using+the+retrograde+horseradish+peroxidase+and+cell+degeneration+techniques.">Somatotopic representation of facial muscles within the facial nucleus of the mouse. A study using the retrograde horseradish peroxidase and cell degeneration techniques.</a> Brain Behav Evol. 24, 144-151 (1984).</li><li>Canh, M. Y., Serpe, C. J., Sanders, V., Jones, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4(+)+T+cell-mediated+facial+motoneuron+survival+after+injury:+Distribution+pattern+of+cell+death+and+rescue+throughout+the+extent+of+the+facial+motor+nucleus.">CD4(+) T cell-mediated facial motoneuron survival after injury: Distribution pattern of cell death and rescue throughout the extent of the facial motor nucleus.</a> Journal of Neuroimmunology. 181, 93-99 (2006).</li><li>Isokawa-Akesson, M., Komisaruk, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difference+in+projections+to+the+lateral+and+medial+facial+nucleus:+anatomically+separate+pathways+for+rhythmical+vibrissa+movement+in+rats.">Difference in projections to the lateral and medial facial nucleus: anatomically separate pathways for rhythmical vibrissa movement in rats.</a> Exp Brain Res. 65, 385-398 (1987).</li><li>Streit, W., Kreutzberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+endogenous+glial+cells+to+motor+neuron+degeneration+induced+by+toxic+ricin.">Response of endogenous glial cells to motor neuron degeneration induced by toxic ricin.</a> The Journal of Comparative Neurology. 268, 248-263 (1988).</li><li>Serpe, C. J., Tetzlaff, J. E., Coers, S., Sanders, V., Jones, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recovery+after+facial+nerve+crush+is+delayed+in+severe+combined+immunodeficient+mice.">Functional recovery after facial nerve crush is delayed in severe combined immunodeficient mice.</a> Brain, Behavior, And Immunity. 16, 808-812 (2002).</li><li>Lal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+facilitates+rat+facial+nerve+recovery+from+a+crush+injury.+Otolaryngology--Head+And+Neck+Surgery.">Electrical stimulation facilitates rat facial nerve recovery from a crush injury. Otolaryngology--Head And Neck Surgery.</a> Official Journal Of American Academy Of Otolaryngology-Head And Neck Surgery. 139, 68-73 (2008).</li><li>Tomov, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Example+of+Neural+Plasticity+Evoked+by+Putative+Behavioral+Demand+and+Early+Use+of+Vibrissal+Hairs+after+Facial+Nerve+Transection.">An Example of Neural Plasticity Evoked by Putative Behavioral Demand and Early Use of Vibrissal Hairs after Facial Nerve Transection.</a> Experimental Neurology. 178, 207-218 (2002).</li><li>Skouras, E., Angelov, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+on+post-transectional+facial+nerve+regrowth+and+functional+recovery+of+paralyzed+muscles+of+the+face+in+rats+and+mice.">Experimental studies on post-transectional facial nerve regrowth and functional recovery of paralyzed muscles of the face in rats and mice.</a> Anatomy (International Journal of Experimental and Clinical Anatomy). 4, 1-27 (2010).</li><li>Xin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-10+within+the+CNS+is+necessary+for+CD4++T+cells+to+mediate+neuroprotection).">IL-10 within the CNS is necessary for CD4+ T cells to mediate neuroprotection).</a> Brain, Behavior, And Immunity. 25, 820-829 (2011).</li><li>Mesnard, N. A., Sanders, V. M., Jones, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+expression+in+the+axotomized+facial+motor+nucleus+of+presymptomatic+SOD1+mice.">Differential gene expression in the axotomized facial motor nucleus of presymptomatic SOD1 mice.</a> The Journal of Comparative Neurology. 519, 3488-3506 (2011).</li><li>Mesnard, N. A., Alexander, T. D., Sanders, V. M., Jones, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+laser+microdissection+in+the+investigation+of+facial+motoneuron+and+neuropil+molecular+phenotypes+after+peripheral+axotomy.">Use of laser microdissection in the investigation of facial motoneuron and neuropil molecular phenotypes after peripheral axotomy.</a> Experimental Neurology. 225, 94-103 (2010).</li><li>Franchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+motor+representation+related+to+facial+nerve+damage+and+regeneration+in+adult+rats.">Changes in motor representation related to facial nerve damage and regeneration in adult rats.</a> Experimental Brain Research. 135, 53-65 (2000).</li><li>Munera, A., Cuestas, D. M., Troncoso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+facial+nerve+lesions+induce+changes+in+the+firing+properties+of+primary+motor+cortex+layer+5+pyramidal+cells.">Peripheral facial nerve lesions induce changes in the firing properties of primary motor cortex layer 5 pyramidal cells.</a> Neuroscience. 223, 140-151 (2012).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hereditary+absence+of+complement+C5+in+adult+mice+influences+Wallerian+degeneration,+but+not+retrograde+responses,+following+injury+to+peripheral+nerve.">Hereditary absence of complement C5 in adult mice influences Wallerian degeneration, but not retrograde responses, following injury to peripheral nerve.</a> Journal of the Peripheral Nervous System. 4, 123-133 (1999).</li><li>Ferri, C., Moore, F., Bisby, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+facial+nerve+injury+on+mouse+motoneurons+lacking+the+p75+low-affinity+neurotrophin+receptor.">Effects of facial nerve injury on mouse motoneurons lacking the p75 low-affinity neurotrophin receptor.</a> Journal of Neurobiology. 34, 1-9 (1997).</li><li>Zhou, R. Y., Xu, J., Chi, F. L., Chen, L. H., Li, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+sensitivity+to+rocuronium+among+orbicularis+oris+muscles+innervated+by+normal+or+damaged+facial+nerves+and+gastrocnemius+muscle+innervated+by+somatic+nerve+in+rats:+combined+morphological+and+functional+analyses.">Differences in sensitivity to rocuronium among orbicularis oris muscles innervated by normal or damaged facial nerves and gastrocnemius muscle innervated by somatic nerve in rats: combined morphological and functional analyses.</a> The Laryngoscope. 122, 1831-1837 (2012).</li><li>Jinno, S., Yamada, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+comparative+anatomy+in+the+axotomy+model+to+identify+distinct+roles+for+microglia+and+astrocytes+in+synaptic+stripping.">Using comparative anatomy in the axotomy model to identify distinct roles for microglia and astrocytes in synaptic stripping.</a> Neuron Glia Biology. 7, 55-66 (2011).</li><li>Jones, K. J., Serpe, C. J., Byram, S. C., Deboy, C. A., Sanders, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+immune+system+in+the+maintenance+of+mouse+facial+motoneuron+viability+after+nerve+injury.">Role of the immune system in the maintenance of mouse facial motoneuron viability after nerve injury.</a> Brain, Behavior, And Immunity. 19, 12-19 (2005).</li><li>Moran, L. B., Graeber, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+facial+nerve+axotomy+model.">The facial nerve axotomy model.</a> Brain research. Brain research. 44, 154-178 (2004).</li><li>Council, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals: Eighth Edition. , (2011).</li><li>Serpe, C. J., Kohm, A. P., Huppenbauer, C. B., Sanders, V., Jones, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exacerbation+of+Facial+Motoneuron+Loss+after+facial+nerve+transection+in+severe+combined+immunodeficient+(scid)+mice.">Exacerbation of Facial Motoneuron Loss after facial nerve transection in severe combined immunodeficient (scid) mice.</a> Neuroscience. 19, (1999).</li><li>Mesnard-Hoaglin, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOD1(G93A)+transgenic+mouse+CD4(+)+T+cells+mediate+neuroprotection+after+facial+nerve+axotomy+when+removed+from+a+suppressive+peripheral+microenvironment.">SOD1(G93A) transgenic mouse CD4(+) T cells mediate neuroprotection after facial nerve axotomy when removed from a suppressive peripheral microenvironment.</a> Brain, Behavior, And Immunity. 40, 55-60 (2014).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+assessment+of+the+perinatal+mouse+facial+nerve+axotomy+model+via+a+subauricular+incision+approach.">Establishment and assessment of the perinatal mouse facial nerve axotomy model via a subauricular incision approach.</a> Experimental Biology And Medicine. 237, 1249-1255 (2012).</li><li>Sharma, N., Moeller, C. W., Marzo, S. J., Jones, K. J., Foecking, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+treatments+enhance+recovery+following+facial+nerve+crush.">Combinatorial treatments enhance recovery following facial nerve crush.</a> The Laryngoscope. 120, 1523-1530 (2010).</li><li>Lieberman, D. M., Jan, T. A., Ahmad, S. O., Most, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+corticosteroids+on+functional+recovery+and+neuron+survival+after+facial+nerve+injury+in+mice.">Effects of corticosteroids on functional recovery and neuron survival after facial nerve injury in mice.</a> Archives of Facial Plastic Surgery. 13, 117-124 (2011).</li><li>Serpe, C. J., Coers, S., Sanders, V. M., Jones, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T,+but+not+CD8++or+B,+lymphocytes+mediate+facial+motoneuron+survival+after+facial+nerve+transection.">CD4+ T, but not CD8+ or B, lymphocytes mediate facial motoneuron survival after facial nerve transection.</a> Brain, Behavior, And Immunity. 17, 393-402 (2003).</li><li>Haulcomb, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axotomy-induced+target+disconnection+promotes+an+additional+death+mechanism+involved+in+motoneuron+degeneration+in+ALS+transgenic+mice.">Axotomy-induced target disconnection promotes an additional death mechanism involved in motoneuron degeneration in ALS transgenic mice.</a> The Journal of Comparative Neurology. , (2014).</li><li>Bauder, A. R., Ferguson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+mouse+sciatic+nerve+crush+and+subsequent+assessment+of+regeneration+by+whole+mount+muscle+analysis.">Reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis.</a> Journal of Visualized Experiments : JoVE. (60), (2012).</li><li>Richner, M., Bjerrum, O. J., Nykjaer, A., Vaegter, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spared+nerve+injury+(SNI)+model+of+induced+mechanical+allodynia+in+mice.">The spared nerve injury (SNI) model of induced mechanical allodynia in mice.</a> Journal of Visualized Experiments : JoVE. (54), (2011).</li></ol>

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...

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 expression1,2. 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 axon bundles. In the crush injury (axonotmesis), the axons are severed, but the perineurium and epineurium are preserved. Complete functional recovery from facial nerve crush occurs in about 11 days because the intact nerve sheath serves as a conduit within which the axons regrow4,5. On the other hand, in the cut injury (neurotmesis), the axons and all 3 connective tissue layers are severed, and the entire distal nerve must regrow to restore musculature innervation. Surgical reconnection of the epineurium is often performed in human patients with nerve transection injuries, however the recovery outcomes are seldom optimal. Further study is required to understand why the nerve fails to regrow to its target and what therapies can be employed to improve and accelerate the regenerative process.
There are many advantages to studying nerve injury using the facial nerve axotomy model. First, the facial nerve axotomy procedure is quick, easy, and highly reproducible; and the resultant paralysis of the facial muscles does not impact vital functions and is well tolerated by the animal. Because this is a cranial nerve injury model, studying the motoneuron cell bodies is simplified because the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons. The population does differ based on the subnuclear pattern within the facial motor nucleus, as there are seven subnuclei each specific to innervating a specific group of muscles, so subnuclear differences in response to axotomy may impact results2,6,7.
A major benefit of the facial nerve injury model is that the unaxotomized side can serve as a paired internal control because the nerve innervation is highly symmetrical and there is no crosstalk between the facial motor nuclei8. Another advantage of using this surgical method is the lack of direct trauma to the CNS or disruption of the blood brain barrier9. Complications such as excessive bleeding and infection are rare with this procedure.
A variety of analyses can be performed to assess the physiologic response to nerve injury. The recovery of the eye blink reflex and whisker activity can be used as a behavioral measure of functional recovery10,11. Video recording of vibrissae activity is currently the most powerful method for detecting recovery of facial nerve innervation12,13. After euthanasia, histological analysis of the brainstem can be performed on the motoneuron cell bodies within the facial motor nucleus. The facial motor nucleus is subdivided into seven subnuclei, each specific to certain facial muscles, allowing for differential examination of responses to injury2,6. Facial motoneurons can be counted to quantify cell survival, or immunohistochemistry can be used to identify biomarkers and specific cell populations14. The facial motor nucleus can be accurately microdissected using laser capture for molecular analysis of the cellular response to nerve injury15,16. Impacts of the facial nerve axotomy can be analyzed in the motor cortex17,18. Also, the nerve can be dissected to study Wallerian degeneration19 or axon regeneration20, and the muscles can be removed to study neuromuscular junctions21. The facial nerve axotomy can also be used to study the accompanying central and peripheral glial cells22, target musculature21, and the immune system response23. Although much has been accomplished in studying the facial nerve axotomy model24, further study of peripheral nerve injury is required because nerve damage is a significant problem for patients and current treatments fail to produce optimal results. This model is a powerful tool for examining the physiologic response to nerve injury and analyzing the effectiveness of nerve regeneration therapies.

<table border="0" cellpadding="0" cellspacing="0" width="980">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:176px;">Catalog Number</td>
			<td style="width:256px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Stereo Microscope</td>
			<td style="width:167px;">Leica</td>
			<td style="width:176px;">M60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Labeling tape</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:176px;">15-952</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:381px;">Vannas-T&#252;bingen Spring Scissors - Straight/Sharp/8.5cm/5mm cutting edge</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">15003-08</td>
			<td>Sterilize before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:381px;">Dumont #5/45 Forceps - Standard tips/Angled 45&#176;/Dumoxel/11cm</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">11251-35</td>
			<td>Sterilize before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Michel Suture Clips - 7.5mm x 1.75 mm</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">12040-01</td>
			<td>Described as &#34;wound clip&#34; in protocol, sterilize before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Hagenbarth Cross Action Wound Clip Applier 5&#34;</td>
			<td style="width:167px;">George Tiemann &#38; Co</td>
			<td style="width:176px;">160-910</td>
			<td>Used to apply wound clip, sterilize before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:381px;">Michel Suture Clip Applicator &#38; Remover - For 7.5 mm Clips</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:176px;">12029-12</td>
			<td>Used to remove wound clip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">0.9% Sodium Chloride Injection, USP</td>
			<td style="width:167px;">Hospira</td>
			<td style="width:176px;">0409-4888-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Betadine, 16 oz, with dispenser</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:176px;">19-027132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">70% Ethanol</td>
			<td style="width:167px;"></td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Glass Bead Sterilizer</td>
			<td style="width:167px;"></td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

facial nerve axotomy in mice: a model to study motoneuron response to injury

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.

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-axotomy14,15,26. Kinetics of motoneuron loss are described in Serp...

Watch this Scientific Journal Video about Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury at JoVE.com

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

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.

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 ...

facial-nerve-axotomy-mice-model-to-study-motoneuron-response-to

Research

JoVE Journal

Medicine

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral procedures to study trigeminal neuropathic pain in rats. To meet the specificity of trigeminal neuropathic pain syndromes, the infraorbital nerve (IoN) is subjected to a chronic constriction injury (CCI) by loosely ligating the nerve. An intra-orbital approach is presented here to expose and ligate the IoN in the orbital cavity. After IoN ligation, rats exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are indicative of persistent pain and mechanical allodynia. Two phases can be defined in the development of the behavioral changes. During the first week following IoN-CCI (phase 1), rats show an increased and asymmetric face grooming activity, i.e., with face wash strokes primarily directed to the nerve-injured IoN territory. A distinction is made between face grooming behavior that is part of a more general body grooming behavior, which remains largely unaffected by IoN-CCI, and face grooming that is neither preceded nor followed by body grooming, which is significantly increased after IoN-CCI. During this period, responsiveness to mechanical stimulation of the IoN territory is reduced. This hyporesponsiveness is abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior (phase 2). The phenomenological similarities between these behavioral alterations and reported signs of facial pain (i.e., responses to noxious stimulation of the face) suggest the presence of dysesthesia/paresthesia and mechanical allodynia in the ligated IoN territory.

Chronic Constriction Injury of the Rat's Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain

This manuscript describes a rodent model to study trigeminal neuropathic pain. The methods described include surgical procedures to perform a chronic constriction injury of the rat&#8217;s infraorbital nerve and the post-surgical behavioral tests to measure the changes in spontaneous and evoked behavior that are indicative of persistent pain and allodynia.

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...