Name: a murine model of cervical spinal cord injury to study post-lesional respiratory neuroplasticity
Uploaded: 2014-05-28T13:00:00
Description: Watch this Scientific Journal Video about A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity at JoVE.com

This work is supported by funding from the European Union Seventh framework Programme (FP7/2007-2013) under grant agreement No. 246556 (European project RBUCE-UP), HandiMedEx allocated by the French Public Investment Board.&#160;Marcel Bonay was supported by the Chancellerie des Universit&#233;s de Paris (Legs Poix), the Fonds de Dotation Recherche en Sant&#233; Respiratoire, and the Centre d&#8217;Assistance Respiratoire &#224; Domicile d&#8217;&#206;le de France (CARDIF)

This protocol was approved by the Ethics committee of the RBUCE-UP chair of Excellence (University of Paris Sud, grant agreement No. 246556) and the Universit&#233; de Versailles Saint-Quentin-en-Yvelines.
1. Preparation of Sterilized Surgical Instruments
<ol>
	<li>Clean the surgical instruments with laboratory detergent.</li>
	<li>Autoclave the instruments prior to surgery.</li>
	<li>In a surgical session, sterilize the tools by placing the tips in a hot bead sterilizer for 10 min at 180 &#...

<ol>
	<li>Frankel, H. 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=Long-term+survival+in+spinal+cord+injury:+a+fifty+year+investigation.">Long-term survival in spinal cord injury: a fifty year investigation.</a> Spinal Cord. 36, 266-274 (1998).</li><li>Ramer, M. S., Harper, G. P., Bradbury, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+spinal+cord+research+-+a+refined+strategy+for+the+International+Spinal+Research+Trust.">Progress in spinal cord research - a refined strategy for the International Spinal Research Trust.</a> Spinal Cord. 38, 449-472 (2000).</li><li>Zimmer, M. B., Nantwi, K., Goshgarian, H. 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S., 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=Respiratory+recovery+following+high+cervical+hemisection.">Respiratory recovery following high cervical hemisection.</a> Respir Physiol Neurobiol. 169, 94-101 (2009).</li><li>Lane, M. A., Lee, K. Z., Fuller, D. D., Reier, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+circuitry+and+respiratory+recovery+following+spinal+cord+injury.">Spinal circuitry and respiratory recovery following spinal cord injury.</a> Respir Physiol Neurobiol. 169, 123-132 (2009).</li><li>Seeds, N. W., Akison, L., Minor, K. <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+plasminogen+activator+in+spinal+cord+remodeling+after+spinal+cord+injury.">Role of plasminogen activator in spinal cord remodeling after spinal cord injury.</a> Respir Physiol Neurobiol. 169, 141-149 (2009).</li><li>Alilain, W. J., Horn, K. P., Hu, H., Dick, T. E., Silver, 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+regeneration+of+respiratory+pathways+after+spinal+cord+injury.">Functional regeneration of respiratory pathways after spinal cord injury.</a> Nature. 475, 196-200 (2011).</li><li>Vinit, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cervical+spinal+cord+injuries+and+respiratory+insufficiency:+a+revolutionary+treatment.">Cervical spinal cord injuries and respiratory insufficiency: a revolutionary treatment.</a> Med Sci (Paris. 28, 33-36 (2012).</li><li>Kastner, A., Gauthier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+rodents+an+appropriate+pre-clinical+model+for+treating+spinal+cord+injury?+Examples+from+the+respiratory+system).">Are rodents an appropriate pre-clinical model for treating spinal cord injury? Examples from the respiratory system).</a> Exp Neurol. 213, 249-256 (2008).</li><li>Vinit, S., Lovett-Barr, M. R., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+hypoxia+induces+functional+recovery+following+cervical+spinal+injury.">Intermittent hypoxia induces functional recovery following cervical spinal injury.</a> Physiol Neurobiol. 169, 210-217 (2009).</li><li>Porter, W. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crossed+phrenic+phenomenon:+a+model+for+plasticity+in+the+respiratory+pathways+following+spinal+cord+injury.">The crossed phrenic phenomenon: a model for plasticity in the respiratory pathways following spinal cord injury.</a> J Appl Physiol. 94, 795-810 (2003).</li><li>Vinit, S., Gauthier, P., Stamegna, J. C., Kastner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+cervical+lateral+spinal+cord+injury+results+in+long-term+ipsilateral+hemidiaphragm+paralysis.">High cervical lateral spinal cord injury results in long-term ipsilateral hemidiaphragm paralysis.</a> J Neurotrauma. 23, 1137-1146 (2006).</li><li>Fuller, D. D., Johnson, S. M., Johnson, R. A., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+cervical+spinal+sensory+denervation+reveals+ineffective+spinal+pathways+to+phrenic+motoneurons+in+the+rat.">Chronic cervical spinal sensory denervation reveals ineffective spinal pathways to phrenic motoneurons in the rat.</a> Neurosci Lett. 323, 25-28 (2002).</li><li>Dougherty, B. J., Lee, K. Z., Lane, M. A., Reier, P. J., Fuller, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+spontaneous+crossed-phrenic+phenomenon+to+inspiratory+tidal+volume+in+spontaneously+breathing+rats.">Contribution of the spontaneous crossed-phrenic phenomenon to inspiratory tidal volume in spontaneously breathing rats.</a> J Appl Physiol. 112, 96-105 (2012).</li><li>Jou, I. 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=Simplified+rat+intubation+using+a+new+oropharyngeal+intubation+wedge.">Simplified rat intubation using a new oropharyngeal intubation wedge.</a> J Appl Physiol. 89, 1766-1770 (2000).</li><li>Fuller, D. 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=Graded+unilateral+cervical+spinal+cord+injury+and+respiratory+motor+recovery.">Graded unilateral cervical spinal cord injury and respiratory motor recovery.</a> Respir Physiol Neurobiol. 165, 245-253 (2009).</li><li>Vinit, S., Windelborn, J. A., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+attenuates+phrenic+long-term+facilitation+following+acute+intermittent+hypoxia.">Lipopolysaccharide attenuates phrenic long-term facilitation following acute intermittent hypoxia.</a> Respir Physiol Neurobiol. 176, 130-135 (2011).</li><li>Ahmad, F., Wang, M. Y., Levi, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia+for+Acute+Spinal+Cord+Injury-A+Review.">Hypothermia for Acute Spinal Cord Injury-A Review.</a> World Neurosurg. , (2013).</li><li>Lovett-Barr, M. R., 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=Repetitive+intermittent+hypoxia+induces+respiratory+and+somatic+motor+recovery+after+chronic+cervical+spinal+injury.">Repetitive intermittent hypoxia induces respiratory and somatic motor recovery after chronic cervical spinal injury.</a> J Neurosci. 32, 3591-3600 (2012).</li><li>Minor, K. H., Akison, L. K., Goshgarian, H. G., Seeds, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury-induced+plasticity+in+the+mouse--the+crossed+phrenic+phenomenon.">Spinal cord injury-induced plasticity in the mouse--the crossed phrenic phenomenon.</a> Exp Neurol. 200, 486-495 (2006).</li><li>Baussart, B., Stamegna, J. C., Polentes, J., Tadie, M., Gauthier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+upper+cervical+spinal+contusion+inducing+a+persistent+unilateral+diaphragmatic+deficit+in+the+adult+rat.">A new model of upper cervical spinal contusion inducing a persistent unilateral diaphragmatic deficit in the adult rat.</a> Neurobiol Dis. 22, 562-574 (2006).</li><li>Golder, F. 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=Breathing+patterns+after+mid-cervical+spinal+contusion+in+rats.">Breathing patterns after mid-cervical spinal contusion in rats.</a> Exp Neurol. 231, 97-103 (2011).</li><li>Lane, M. 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=Respiratory+function+following+bilateral+mid-cervical+contusion+injury+in+the+adult+rat.">Respiratory function following bilateral mid-cervical contusion injury in the adult rat.</a> Exp Neurol. 235, 197-210 (2012).</li><li>Vinit, S., 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=Axotomized+bulbospinal+neurons+express+c-Jun+after+cervical+spinal+cord+injury.">Axotomized bulbospinal neurons express c-Jun after cervical spinal cord injury.</a> Neuroreport. 16, 1535-1539 (2005).</li><li>Guenther, C. H., Windelborn, J. A., Tubon, T. C., Yin, J. C., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+atypical+PKC+expression+and+activity+in+the+phrenic+motor+nucleus+following+cervical+spinal+injury.">Increased atypical PKC expression and activity in the phrenic motor nucleus following cervical spinal injury.</a> Exp Neurol. 234, 513-520 (2012).</li><li>Mantilla, C. B., Gransee, H. M., Zhan, W. Z., Sieck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motoneuron+BDNF/TrkB+signaling+enhances+functional+recovery+after+cervical+spinal+cord+injury.">Motoneuron BDNF/TrkB signaling enhances functional recovery after cervical spinal cord injury.</a> Exp Neurol. 247, 101-109 (2013).</li><li>Vinit, S., Darlot, F., Aoulaiche, H., Boulenguez, P., Kastner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+expression+of+c-Jun+and+HSP27+in+axotomized+and+spared+bulbospinal+neurons+after+cervical+spinal+cord+injury.">Distinct expression of c-Jun and HSP27 in axotomized and spared bulbospinal neurons after cervical spinal cord injury.</a> J Mol Neurosci. 45, 119-133 (2011).</li><li>Windelborn, J. A., Mitchell, G. 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Technical Difficulties of Making the C2 Injury Model
The C2 injury murine model is an interesting tool to study respiratory post-lesional neuroplasticity. However, the steps needed to produce a reproducible and reliable model are numerous and each one could impact on the outcome of the study. For example, during the intubation process, extreme care is to be taken since the orotracheal tube can produce an inflammation of the trachea, which can lead to diverse complications such...

Spinal cord trauma is a common injury observed in the human population with dramatic incidences, such as permanent paralysis. However, the severity of the injury depends on the level and the extent of the initial trauma. Respiratory failure is the leading cause of mortality following upper cervical spinal cord injury (SCI)1. Currently, the only therapeutic treatment is to place the patient under ventilatory assistance. Since few patients can be weaned off the ventilatory assistance2, due to spontaneous recovery which occurs with post-lesional delay, the need to develop new innovative non-invasive therapeutics is urgent3. Having a good standardized pre-clinical model to investigate the effect of a cervical SCI on respiratory insufficiency and therefore, to study the application of putative therapeutic strategies, is essential.
In this technical article, we describe a specific pre-clinical murine model of respiratory impairment induced by a partial cervical SCI at the C2 level. This model is currently used by several laboratories around the world (for reviews: 4-13). However, slight differences in the surgical procedure can be observed among the different investigators to generate this particular cervical injury murine model. The effect of a C2 SCI on the respiratory output was first described in 1895 by Porter14. A cervical hemisection induces a deafferentation of the phrenic motoneurons from their central drive (located in the rVRG in the brainstem, Figure 1A) on the ipsilateral side of injury, leading to a silent phrenic nerve activity and the subsequent diaphragm paralysis. The contralateral side remains intact and allows the animal to survive. Unlike different SCI located in a lower spinal segment (for example a contusive injury at C4 level15), the integrity of the phrenic motoneuron nucleus on both side is preserved. After a cervical C2 injury, some spontaneous activity can be observed on the ipsilateral side (phrenic and diaphragm) due to an activation of contralateral silent synaptic pathways which crossed the spinal midline at the segmental level C3-C6 (Crossed phrenic pathways, CPP, Figure 1B). The activation of the CPP, which is, by definition, a C2 hemisection combined with a contralateral phrenicotomy which induce an ipsilateral partial phrenic nerve recovery, can occur from hours to weeks post-injury16-18. The real beneficial effect of this CPP pathway on the respiratory recovery is limited19 and further investigation and treatment should be developed to improve the magnitude of spontaneous restoration3.
This protocol provides a powerful type of pre-clinical murine model to study respiratory post-lesional plasticity at various levels (respiratory physiology from pre and phrenic motoneurons, interneurons, molecular and cellular, locomotion of the front limb for example) as well as a model to test invasive and non-invasive therapeutic strategies aimed to improve the respiratory and locomotor recovery following C2 partial cervical spinal cord injury.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:572px;">Animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Male Sprague Dawley Rat</td>
			<td>Janvier</td>
			<td></td>
			<td>225-250g</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Surgical Instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Student Dumont #5 forceps</td>
			<td>Fine Science Tool</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Student Standard Pattern Forceps</td>
			<td>Fine Science Tool</td>
			<td>91100-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mayo-Stille Scissors</td>
			<td>Fine Science Tool</td>
			<td>14013-15</td>
			<td>Curved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Student Vannas Spring Scissors</td>
			<td>Fine Science Tool</td>
			<td>91500-09</td>
			<td>Straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors - 8 mm Blades</td>
			<td>Fine Science Tool</td>
			<td>15025-10</td>
			<td>Straight Blunt/Blunt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Friedman Pearson Rongeur</td>
			<td>Fine Science Tool</td>
			<td>16121-14</td>
			<td>Curved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Knife - Fine Tip</td>
			<td>Fine Science Tool</td>
			<td>10055-12</td>
			<td>Straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olsen-Hegar Needle Holder</td>
			<td>Fine Science Tool</td>
			<td>12002-14</td>
			<td>Serrated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weitlaner-Locktite Retractor</td>
			<td>Fine Science Tool</td>
			<td>17012-11</td>
			<td>2x3 Blunt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absorbable surgical sutures</td>
			<td>Centravet</td>
			<td>BYO001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hot Bead Steriliser</td>
			<td>Fine Science Tool</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Catheter&#160;</td>
			<td>Centravet</td>
			<td>CAT188</td>
			<td>16 gauge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laryngoscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guide wire</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laryngeal mirror</td>
			<td>Centravet</td>
			<td>MIR011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactated Ringers</td>
			<td>Centravet</td>
			<td>RIN020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe</td>
			<td>Centravet</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle</td>
			<td>Centravet</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">O2</td>
			<td>Air Liquid</td>
			<td>I1001M20R2A001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">683 RodentT Ventilator 115/230V</td>
			<td>Havard Apparatus</td>
			<td>55-0000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stand-Alone Vaporizer</td>
			<td>WPI</td>
			<td>EZ-155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thin line heated bed</td>
			<td>WPI</td>
			<td>EZ-211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Air canister</td>
			<td>WPI</td>
			<td>EZ-258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Drugs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carprofen</td>
			<td>Centravet</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rimadyl</td>
			<td>Centravet</td>
			<td>RIM011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine</td>
			<td>Centravet</td>
			<td>BUP001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Baytril</td>
			<td>Centravet</td>
			<td>BAY001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dexmedetomidine</td>
			<td>Centravet</td>
			<td>DEX010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Atipamezole</td>
			<td>Centravet</td>
			<td>ANT201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine Solution</td>
			<td>Centravet</td>
			<td>VET002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Centravet</td>
			<td>VET066</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Extent of Injury
The success and the reproducibility of this particular experimental model are dependent on the experience of each manipulator/surgeon. The subsequent amount of respiratory recovery (phrenic nerve activity and diaphragm activity) following a C2 injury is correlated with the remaining ventrolateral spared white matter21. Since the injury is &#8220;handmade&#8221; and requires some practice from the surgeon, the extent of each injury has to be checked ...

Watch this Scientific Journal Video about A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity at JoVE.com

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

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 ...

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Research

JoVE Journal

Medicine

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...