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 ...

<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 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 ...

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Research

JoVE Journal

Neuroscience

11.7K visualizações.  University of Rouen.  Células olfativas ensheathing (OECO) são células da crista neural que permitem o crescimento dos neurônios olfativos primários. Esta propriedade específica pode ser utilizado para transplantação celular. Apresentamos aqui um modelo de transplante celular baseado na utilização de OECs em um modelo de lesão do nervo laríngeo. 