The authors would like to acknowledge ADIR (Aide &#224; Domicile aux Insuffisants Respiratoires) and Fondation de l&#8217;Avenir for their financial support and to Dr. Fanie Barnab&#233;-Heider for editing the manuscript.

1. Primary Cultures of Olfactory Mucosa and Olfactory Bulbs
<ol>
	<li>Prior to dissection
	<ol>
		<li>Make medium, for 50 ml, by adding 44 ml of calcium-free Dulbecco's Modified Eagle's/Ham's F12 medium (DMEM/F12), supplemented with 5 ml of Fetal Bovine Serum (FBS) and 1 ml of penicillin/streptomycin.</li>
		<li>Coat 75 cm2 flasks with poly-L-lysine (50 &#956;g/ml, 1.5 &#956;g/cm2), 1 hr at room temperature.</li>
		<li>Rinse flasks with PBS 1x.</li>
		<li>Store the coated flasks in the fridge for up to 1 week.</li>
	</ol>
	</li>
	<li>Dissection
	<ol>
		<li>Any method of euthanasia must be approved in advance by the institution&#39;s animal care and use committee and carried out by qualified personnel.</li>
		<li>Choose inbred rats such as Fischer rats.</li>
		<li>After the rat has been confirmed to have entered deep anesthesia with sodium pentobarbital or other injectable forms of anesthesia such as ketamine/xylazine, decapitate him and remove the skin.</li>
		<li>Open the skull from the anterior to the posterior part.</li>
		<li>Remove the OB taking care to avoid the meninges.</li>
		<li>In the same rat, open the nasal cavity sagittaly.</li>
		<li>Remove the OM taking care to avoid the respiratory mucosa. Note that the OM epithelium can be easily identified by its yellowish color.</li>
		<li>Further detailed protocols about dissection of OM can be find in a previous <a href="https://www.jove.com/video/2762/isolating-nasal-olfactory-stem-cells-from-rodents-or-humans">J. Vis. Exp</a>. publication13.</li>
	</ol>
	</li>
	<li>Culture
	<ol>
		<li>Place OB in Hank's buffered salt solution (HBSS) containing 0.1% trypsin for 45 min at 37 &#176;C.</li>
		<li>Place OM in HBSS containing 0.05% collagenase A for 45 min at 37 &#176;C.</li>
		<li>Stop enzymatic digestion by adding 2 ml of warm medium.</li>
		<li>Wash the cells with medium and centrifuge them at 300 x g for 3 min.</li>
		<li>Triturate samples of 2 ml of medium using a P1000 pipette until a homogenous cell suspension is obtained.</li>
		<li>Plate the cells in the precoated flasks (5-10 x 106 cells/flask) with 25 ml of warm medium.</li>
		<li>Incubate and culture the cells at 37 &#176;C with 5% CO2.</li>
		<li>Change half of the medium every 2 days.</li>
		<li>After 6-8 days in vitro, check the proportion of p75 positive cells on small cell samples, assumed to be OECs in rodents, by flow cytometry or by immunocytochemistry. For these analyses, other markers can be studied such as GFAP and S100&#946;.</li>
	</ol>
	</li>
	<li>Flow cytometry analysis
	<ol>
		<li>Collect the cells as described in "cellular transplantation" (section 2.2 below).</li>
		<li>Incubate them with p75 primary antibody (1:100) during 15 min at 4 &#176;C.</li>
		<li>Wash the cells with 2 ml of PBS-EDTA and centrifuge them at 300 x g for 3 min.</li>
		<li>Incubate the cells with PE conjugated anti-mouse secondary antibody during 15 min at 4 &#176;C in the dark (1:200).</li>
		<li>Wash the cells with 2 ml of PBS-EDTA and centrifuge them at 300 x g for 3 min.</li>
		<li>Resuspend the cells in 500 &#956;l of PBS-EDTA.</li>
		<li>Analyze 10-30 x 103 cells with flow-cytometer.</li>
	</ol>
	</li>
	<li>Immunocytochemistry analysis
	<ol>
		<li>Plate the cells in 6-well plates precoated for 48 hr as described in "culture" (section 1.1.2 below).</li>
		<li>Wash the cells with PBS.</li>
		<li>Fix the cells with PFA 4% for 15 min at RT.</li>
		<li>Wash the cells with PBS.</li>
		<li>Incubate the cells with PBS/Triton X100 (0.1%) for 15 min at RT.</li>
		<li>Wash the cells with PBS.</li>
		<li>Incubate the cells with primary antibodies (1:200): p75, S100&#946; and GFAP overnight at 4 &#176;C.</li>
		<li>Wash the cells with PBS.</li>
		<li>Incubate the cells with the secondary antibodies (1:500) during 1 hr at RT in darkness.</li>
		<li>Wash the cells with PBS.</li>
		<li>Incubate the cells with Hoechst 10 min at RT in darkness.</li>
		<li>Wash the cells with PBS.</li>
		<li>Analyze the cells using a fluorescent microscope.</li>
	</ol>
	</li>
	<li>GFP labeling cells
	<ol>
		<li>Five days before transplantation, infect OB and OM cultures overnight with lentiviral vector harboring enhanced GFP (multiplicity of infection: 20).</li>
		<li>Before transplantation check on small samples the rate of GFP positive cells by flow-cytometry as described previously in "culture" (section 1.1).</li>
	</ol>
	</li>
</ol>
2. Surgery and Transplantation
<ol>
	<li>Recurrent nerve anastomosis
	<ol>
		<li>All experiments with animals must be approved in advance by the institution&#39;s animal care and use committee and carried out by qualified personnel.</li>
		<li>Prior to surgery autoclave all instruments and maintain sterility throughout the surgical procedures.</li>
		<li>Anesthetize rat by intraperitoneal injection of ketamine hydrochloride (12.5 mg/kg) and chlorpromazine hydrochloride (0.625 mg/kg). Assess adequacy of anesthesia by toe pinch prior to surgery.</li>
		<li>Place the rat in dorsal decubitus.</li>
		<li>Perform a 2 cm vertical medium cervical cutaneous incision with a scalpel.</li>
		<li>Perform a vertical incision of the infrahyoid muscles following the medial white line.</li>
		<li>Expose larynx by using autostatic retractor between infrahyoid muscles.</li>
		<li>Expose the RLN.</li>
		<li>Under microscopic control, cut the nerve fully with microscissors at the level of the seventh tracheal ring.</li>
		<li>Perform anastomosis using one point of 11.0 suture under microscopic control. This surgery leads to a specific motor denervation of the larynx.</li>
	</ol>
	</li>
	<li>Cellular transplantation
	<ol>
		<li>Just before transplantation, remove the cells from the dishes using 0.05% trypsin EDTA for 5 min at 37 &#176;C.</li>
		<li>Stop enzymatic digestion by adding 2 ml of warm medium.</li>
		<li>Centrifuge at 300 x g for 3 min.</li>
		<li>Under a microscope count the cells using a hemocytometer and adjust the cellular concentration according to specific protocol. In this model, desired concentrations were between 1.2-6 x 106 cells in 30 &#956;l of DMEM/F12.</li>
		<li>Make cellular preparation with 1:2 mix (30 and 60 &#956;l in this experiment) of DMEM/F12-Matrige l in 1.5 ml tube before grafting and place it on ice. To avoid unspecific effects, use growth factor reduced Matrigel.</li>
		<li>Just prior to transplantation hold the tube in your hand for a few seconds to warm up the Matrigel-medium mix.</li>
		<li>Depose the mix on the anastomosis site using a pipette and wait until the mixture self-assembles on the injured nerve.</li>
		<li>Close the muscles and skin with a 4.0 suture.</li>
		<li>House the rats individually and keep under a heating lamp for 24 hr.</li>
	</ol>
	</li>
</ol>
3. Evaluation
<ol>
	<li>Laryngoscopy
	<ol>
		<li>Anesthetize rats by intraperitoneal injection of ketamine hydrochloride (12.5 mg/kg) and chlorpromazine hydrochloride (0.625 mg/kg). Confirm rats are anesthetized with a toe pinch prior to proceeding with protocol.</li>
		<li>Place the rat in dorsal decubitus.</li>
		<li>Insert a 30&#176; videolaryngoscope adjusted to provide the best view of the larynx. Determine anatomic landmarks to reproduce the same view for each recording.</li>
		<li>Record vocal cord movements using the videolaryngoscope (5-10 sequences/animal). For each animal select three sequences of successive maximal adduction and maximal abduction.</li>
		<li>Analyze laryngeal function using image analysis software. Determine absolute angular movement by measuring the difference between movement of maximal abduction and maximal adduction of the vocal cords. Determine dynamic score by measuring the movement amplitude. Determine functional score by measuring the synkinesis and paradoxal movements.</li>
	</ol>
	</li>
	<li>Electromyography (EMG)
	<ol>
		<li>Place an electrode on the hamstring muscles of the rat for electrical isolation.</li>
		<li>Expose larynx and trachea by opening skin and muscles as described previously in "recurrent nerve anastomosis" (section 2.1).</li>
		<li>Expose the cricothyroid cartilage.</li>
		<li>Open the cricothyroid cartilage to expose the posterior cricoarytenoid (PCA) muscle using microscissors.</li>
		<li>Introduce a monopolar needle electrode (38 x 0.45 mm) in the PCA muscle.</li>
		<li>Record the electrical muscular activity of the PCA muscle using an acquisition system during spontaneous ventilation.</li>
		<li>Analyze the muscular activity in terms of richness and synchronization with the respiration. To analyze EMGs, assign a qualitative score from 0-3 using the following scale: 0: unrhymed tracing, without increase during inspiration, 1: rhymed tracing with inspiratory increasing, but poor tracing (neurogen), 2: rhymed tracing with richer activity, 3: rhymed tracing, very rich, constituting an interference pattern, similar to a maximal intentional activity.</li>
		<li>Expose the vagus nerve to measure latency and potential duration. The vagus nerve is chosen as the site of stimulation because RLN can be damaged during stimulation due to its small size.</li>
		<li>Stimulate the vagus nerve with an electrode.</li>
		<li>Record electrical signals in the PCA muscle. Measure latency and potential duration. If possible use an acquisition module to record muscular activity, latency and potential duration such as Powerlab system.</li>
	</ol>
	</li>
	<li>Histology
	<ol>
		<li>Euthanize the animal using pentobarbital overdose.</li>
		<li>Remove the distal stump of RLN under microscopic control. The distal part of RLN is defined as the part between anastomosis and larynx. Note that it is possible to find the anastomosis site several weeks after surgery because the 11.0 suture is a nonabsorbable suture.</li>
		<li>Place the distal portion of RLN in 2% glutaraldehyde with 0.1 M phosphate (pH=7.3) for 2 hr at 4 &#176;C.</li>
		<li>Carefully orient the samples to realize coronal sections.</li>
		<li>Wash samples in phosphate buffer.</li>
		<li>Cut in smaller segments (3-4 mm length) and postfix for 60 min at room temperature in 1% cacodylate buffered OsO4 and dehydrated with ethanol.</li>
		<li>Orient samples in silicone molds and embedded in resins consisting in POLYBED 812, DDSA and MNA, to which was added 2% of the accelerator BDMA.</li>
		<li>Make semi-thin transverse sections (1&#956;m-thick) using a Pyramitome Ultramicrotomy System.</li>
		<li>Stain with 0.1% Toluidine Blue in 1% sodium tetraborate for 75 sec at 70 &#176;C.</li>
		<li>Analyze RLN samples with a counting analysis system. Count the number of fibers and measure myelinated nerve fiber profiles by determining the outer and inner boundary of the myelin sheath.</li>
	</ol>
	</li>
	<li>Transplantation
	<ol>
		<li>Prepare cells for transplantation and transplant the cells as previously described in "cellular transplantation" (section 2.2).</li>
		<li>Analysis</li>
		<li>Euthanize the animal using pentobarbital overdose. Note that fixation of the animals can be performed using intracardiac injection of PFA 4%.</li>
		<li>Remove around 2 cm of RLN. Use proximal and distal parts of the RLN.</li>
		<li>Fix RLN sample in liquid nitrogen.</li>
		<li>Store samples at -80 &#176;C.</li>
		<li>Cut samples longitudinally using cryostat (10-20 &#956;m thick).</li>
		<li>Analyze samples under fluorescent microscope to detect GFP positive cells. Note that anti-GFP antibody can be used to increase fluorescence emission and to perform costainings.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Barraud, P., 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=Neural+crest+origin+of+olfactory+ensheathing+glia.">Neural crest origin of olfactory ensheathing glia.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 21040-21045 (2010).</li><li>Guerout, N., 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=Comparative+gene+expression+profiling+of+olfactory+ensheathing+cells+from+olfactory+bulb+and+olfactory+mucosa.">Comparative gene expression profiling of olfactory ensheathing cells from olfactory bulb and olfactory mucosa.</a> Glia. 58, 1570-1580 (1002).</li><li>Honore, 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=Isolation,+characterization,+and+genetic+profiling+of+subpopulations+of+olfactory+ensheathing+cells+from+the+olfactory+bulb.">Isolation, characterization, and genetic profiling of subpopulations of olfactory ensheathing cells from the olfactory bulb.</a> Glia. 60, 404-413 (2012).</li><li>Franssen, E. H., de Bree, F. M., Verhaagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+glia:+their+contribution+to+primary+olfactory+nervous+system+regeneration+and+their+regenerative+potential+following+transplantation+into+the+injured+spinal+cord.">Olfactory ensheathing glia: their contribution to primary olfactory nervous system regeneration and their regenerative potential following transplantation into the injured spinal cord.</a> Brain Res. Rev. 56, 236-258 (2007).</li><li>Lakatos, A., Franklin, R. J., Barnett, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+and+Schwann+cells+differ+in+their+in+vitro+interactions+with+astrocytes.">Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes.</a> Glia. 32, 214-225 (2000).</li><li>Woodhall, E., West, A. K., Chuah, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+olfactory+ensheathing+cells+express+nerve+growth+factor,+brain-derived+neurotrophic+factor,+glia+cell+line-derived+neurotrophic+factor+and+their+receptors.">Cultured olfactory ensheathing cells express nerve growth factor, brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor and their receptors.</a> Brain Res. Mol. Brain Res. 88, 203-213 (2001).</li><li>Dombrowski, M. A., Sasaki, M., Lankford, K. L., Kocsis, J. D., Radtke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+and+nodal+formation+of+regenerated+peripheral+nerve+fibers+following+transplantation+of+acutely+prepared+olfactory+ensheathing+cells.">Myelination and nodal formation of regenerated peripheral nerve fibers following transplantation of acutely prepared olfactory ensheathing cells.</a> Brain Res. 1125, 1-8 (2006).</li><li>Guerout, N., 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=Transplantation+of+olfactory+ensheathing+cells+promotes+axonal+regeneration+and+functional+recovery+of+peripheral+nerve+lesion+in+rats.">Transplantation of olfactory ensheathing cells promotes axonal regeneration and functional recovery of peripheral nerve lesion in rats.</a> Muscle Nerve. 43, 543-551 (2011).</li><li>Guntinas-Lichius, O., 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=Transplantation+of+olfactory+ensheathing+cells+stimulates+the+collateral+sprouting+from+axotomized+adult+rat+facial+motoneurons.">Transplantation of olfactory ensheathing cells stimulates the collateral sprouting from axotomized adult rat facial motoneurons.</a> Exp. Neurol. 172, 70-80 (2001).</li><li>Makoukji, 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=Lithium+enhances+remyelination+of+peripheral+nerves.">Lithium enhances remyelination of peripheral nerves.</a> Proc. Natl. Acad. Sci U.S.A. 109, 3973-3978 (2012).</li><li>Guerout, N., 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=Co-transplantation+of+olfactory+ensheathing+cells+from+mucosa+and+bulb+origin+enhances+functional+recovery+after+peripheral+nerve+lesion.">Co-transplantation of olfactory ensheathing cells from mucosa and bulb origin enhances functional recovery after peripheral nerve lesion.</a> PloS one. 6, 22816 (2011).</li><li>Paviot, 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=Efficiency+of+laryngeal+motor+nerve+repair+is+greater+with+bulbar+than+with+mucosal+olfactory+ensheathing+cells.">Efficiency of laryngeal motor nerve repair is greater with bulbar than with mucosal olfactory ensheathing cells.</a> Neurobiol. Dis. 41, 688-694 (2011).</li><li>Girard, S. 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=Isolating+nasal+olfactory+stem+cells+from+rodents+or+humans.">Isolating nasal olfactory stem cells from rodents or humans.</a> J. Vis. Exp. , (2011).</li><li>Nash, H. H., Borke, R. C., Anders, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+method+of+purification+for+establishing+primary+cultures+of+ensheathing+cells+from+the+adult+olfactory+bulb.">New method of purification for establishing primary cultures of ensheathing cells from the adult olfactory bulb.</a> Glia. 34, 81-87 (2001).</li><li>Bianco, J. I., Perry, C., Harkin, D. G., Mackay-Sim, A., Feron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin+3+promotes+purification+and+proliferation+of+olfactory+ensheathing+cells+from+human+nose.">Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose.</a> Glia. 45, 111-123 (2004).</li><li>Paviot, A., Bon-Mardion, N., Duclos, C., Marie, J. P., Guerout, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Although+olfactory+ensheathing+cells+have+remarkable+potential+to+sustain+nerve+regeneration,+they+cannot+be+applied+to+a+severe+vagus+nerve+section/resection+model.">Although olfactory ensheathing cells have remarkable potential to sustain nerve regeneration, they cannot be applied to a severe vagus nerve section/resection model.</a> Muscle Nerve. 43, 919-920 (2011).</li></ol>

The authors have nothing to disclose.

The techniques presented here make OECs a useful model to study cellular transplantations in peripheral nerve injury models. The cell culture protocol is relatively straightforward and can be easily carried out. On the other hand, surgical procedures, in particular section/anastomosis of the RLN, require experience and must be carried out by qualified personnel.
The procedures described in this protocol highlight important factors to focus on in order to obtain the best results possible. First...

The primary olfactory system is composed of two distinct parts; the olfactory mucosa (OM) in peripheral nervous system and the olfactory bulb (OB) in the central nervous system. The primary olfactory system is characterized by the capacity of the primary olfactory neurons (PON) to self-renew throughout life in mammal species. This ability is made possible due to the presence of neural stem cells in the OM. PON growth and regrowth from OM to OB is facilitated by specialized glial cells called olfactory ensheathing cells (OECs). OECs are neural crest cells which create a favorable microenvironment for neurogenesis of the PON from OM to OB1. Thereby, OECs can be found in OM and in OB constituting different subpopulations of cells2,3. The different properties of OECs have lead scientists to use them for cellular transplantations in several nervous system lesion paradigms4. Indeed, OECs produce growth factors, reduce glial scaring, promote axonal regrowth, and can freely intermingle with astrocytes5,6. However, the vast majority of these studies are based on spinal cord injury (SCI); few of them have used OECs after peripheral nerve injury (PNI)7,8.
Although the peripheral nervous system has a great capacity to regenerate after nerve injury, complete sections induce aberrant axonal regrowth. Indeed, after complete transections of the facial or the recurrent laryngeal nerves (RLN) misrouted axons cause muscular cocontractions called synkinesis. Therefore it is of primary importance to propose a model of PNI to not only quantify axonal regrowth but also to evaluate the efficiency of the muscular contractions. In the literature the most common model described is based on facial nerve lesion9,10. In this model, functional evaluations are based on the recovery of whisker movements10. However it is complicated to demonstrate the efficiency of the movements and to discriminate the muscular cocontractions phenomena. We propose here a model based on a RLN lesion. This model allows the evaluation of not only axonal regrowth and movements of the vocal cords but also the efficiency and functionality of these movements after cellular transplantations11,12. This protocol provides a step by step procedure to culture and transplant OECs from OM and OB in an RLN section/anastomosis model and to evaluate animals after surgery.

<table border="0" cellpadding="0" cellspacing="0" width="591">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">DMEM/F12</td>
			<td style="width:155px;">Invitrogen</td>
			<td style="width:197px;">E3521T</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Invitrogen</td>
			<td>E3387M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicilin/streptomycin</td>
			<td>Invitrogen</td>
			<td>1152-8876</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS</td>
			<td>Invitrogen</td>
			<td>M3467Y</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>M3513P</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cacodylate</td>
			<td>Merck</td>
			<td>1.03256.0100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DDSA</td>
			<td>BIOVALLEY</td>
			<td>00563-450</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MNA</td>
			<td>BIOVALLEY</td>
			<td>00886-450</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BDMA</td>
			<td>BIOVALLEY</td>
			<td>00141-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">POLYBED 812</td>
			<td>BIOVALLEY</td>
			<td>08791-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE anti-mouse&#160;</td>
			<td>BD Bioscience&#160;</td>
			<td>550589</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel GFR</td>
			<td>BD Bioscience&#160;</td>
			<td>356231</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase A</td>
			<td>Roche</td>
			<td>10103586001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti P75</td>
			<td>Chemicon</td>
			<td>MAB 365</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">11.0 Wire</td>
			<td>Ethicon&#160;</td>
			<td>FG 2881</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Toluidine Blue&#160;</td>
			<td>RAL DIAGNOSTICS</td>
			<td>361590-0025</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Sigma&#160;</td>
			<td>Sigma 2-16PK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator&#160;</td>
			<td>Thermo scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminar flow hood</td>
			<td>Faster</td>
			<td style="width:197px;">BH-EN 2003 S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow cytometer</td>
			<td>BD Bioscience&#160;</td>
			<td>FACSCalibur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Videolaryngoscope</td>
			<td>Karl Storz Endoskope</td>
			<td>Telecam SL NLSC 20212120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acquisition system</td>
			<td>ADInstruments</td>
			<td>Powerlab system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyramitome Ultramicrotomy System&#160;</td>
			<td style="width:155px;">Leica</td>
			<td>Ultracut S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image analysis system</td>
			<td>Explora Nova</td>
			<td>Mercator</td>
		</tr>
	</tbody>
</table>

transplantation of olfactory ensheathing cells to evaluate functional recovery after peripheral nerve injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Illustrations with control and reinnervated (section/anastomosed) animals have been chosen as results can vary depending on the cellular transplantations performed (OM, OB or OM+OB).
Cell culture 
	The cells adhere rapidly to the plastic surface and became segregated into parallel swaths of cells that are elongated or tapering, triangular, multipolar, or spindle-shaped (Figures 1A and 1B). After 8 days in vitro flow cytometry analysis show...

Watch this Scientific Journal Video about Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury at JoVE.com

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

transplantation-olfactory-ensheathing-cells-to-evaluate-functional

Research

JoVE Journal

Neuroscience

11.7K Views.  University of Rouen. Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Video: Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask gait analysis consisted of a single walking task (Task 0), a simple motor dual-task (water-holding, Task 1), and a complex motor dual-task (crossing obstacles, Task 2). The task of crossing obstacles was considered to be equivalent to the combination of a simple walking task and a complex motor task as it involved more nervous system, skeletal movement, and cognitive resources. To eliminate heterogeneity in the results of the gait analysis of the stroke patients, the dual-task gait cost values were calculated for various kinematic parameters. The major differences were observed in the proximal joint angles, especially in the angles of the trunk, pelvis, and hip joints, which were significantly larger in the dual motor tasks than in the single walking task. This research protocol aims to provide a basis for the clinical diagnosis of gait function and an in-depth study of motor control in stroke patients with motor control deficits through the analyses of dual-motor walking tasks.

This paper presents a protocol specifically for dual motor task gait analysis in stroke patients with motor control deficits.

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask ...