Name: Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury (Video) | JoVE Journal
Uploaded: 2014-02-23T16:00:00.000+00:00
Duration: 10 min 33 s
Description: Learn olfactory ensheathing cell transplantation for peripheral nerve regeneration using a rat laryngeal injury model. This method evaluates functional recovery through laryngoscopy, electromyography, and histological analysis.

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5 minutes

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/pubmed?term=((Neural+crest+origin+of+olfactory+ensheathing+glia%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">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/pubmed?term=((Comparative+gene+expression+profiling+of+olfactory+ensheathing+cells+from+olfactory+bulb+and+olfactory+mucosa%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((Isolation%2C+characterization%2C+and+genetic+profiling+of+subpopulations+of+olfactory+ensheathing+cells+from+the+olfactory+bulb%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((Olfactory+ensheathing+glia%3A+their+contribution+to+primary+olfactory+nervous+system+regeneration+and+their+regenerative+potential+following+transplantation+into+the+injured+spinal+cord%5BTitle%5D)+AND+%22Brain+Res.+Rev%22%5BJournal%5D)">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/pubmed?term=((Olfactory+ensheathing+cells+and+Schwann+cells+differ+in+their+in+vitro+interactions+with+astrocytes%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((Cultured+olfactory+ensheathing+cells+express+nerve+growth+factor%2C+brain-derived+neurotrophic+factor%2C+glia+cell+line-derived+neurotrophic+factor+and+their+receptors%5BTitle%5D)+AND+%22Brain+Res.+Mol.+Brain+Res%22%5BJournal%5D)">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/pubmed?term=((Myelination+and+nodal+formation+of+regenerated+peripheral+nerve+fibers+following+transplantation+of+acutely+prepared+olfactory+ensheathing+cells%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">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/pubmed?term=((Transplantation+of+olfactory+ensheathing+cells+promotes+axonal+regeneration+and+functional+recovery+of+peripheral+nerve+lesion+in+rats%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">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/pubmed?term=((Transplantation+of+olfactory+ensheathing+cells+stimulates+the+collateral+sprouting+from+axotomized+adult+rat+facial+motoneurons%5BTitle%5D)+AND+%22Exp.+Neurol%22%5BJournal%5D)">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/pubmed?term=((Lithium+enhances+remyelination+of+peripheral+nerves%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci+U.S.A%22%5BJournal%5D)">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/pubmed?term=((Co-transplantation+of+olfactory+ensheathing+cells+from+mucosa+and+bulb+origin+enhances+functional+recovery+after+peripheral+nerve+lesion%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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/pubmed?term=((Efficiency+of+laryngeal+motor+nerve+repair+is+greater+with+bulbar+than+with+mucosal+olfactory+ensheathing+cells%5BTitle%5D)+AND+%22Neurobiol.+Dis%22%5BJournal%5D)">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/pubmed?term=((Isolating+nasal+olfactory+stem+cells+from+rodents+or+humans%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">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/pubmed?term=((New+method+of+purification+for+establishing+primary+cultures+of+ensheathing+cells+from+the+adult+olfactory+bulb%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((Neurotrophin+3+promotes+purification+and+proliferation+of+olfactory+ensheathing+cells+from+human+nose%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((Although+olfactory+ensheathing+cells+have+remarkable+potential+to+sustain+nerve+regeneration%2C+they+cannot+be+applied+to+a+severe+vagus+nerve+section%2Fresection+model%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">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><tbody><tr><td>DMEM/F12</td><td>Invitrogen</td><td>E3521T</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>E3387M</td><td></td></tr><tr><td>Penicilin/streptomycin</td><td>Invitrogen</td><td>1152-8876</td><td></td></tr><tr><td>HBSS</td><td>Invitrogen</td><td>M3467Y</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>M3513P</td><td></td></tr><tr><td>Cacodylate</td><td>Merck</td><td>1.03256.0100</td><td></td></tr><tr><td>DDSA</td><td>Biovalley</td><td>00563-450</td><td></td></tr><tr><td>MNA</td><td>Biovalley</td><td>00886-450</td><td></td></tr><tr><td>BDMA</td><td>Biovalley</td><td>00141-100</td><td></td></tr><tr><td>Polybed 812</td><td>Biovalley</td><td>08791-500</td><td></td></tr><tr><td>PE anti-mouse&amp;nbsp;</td><td>BD Bioscience</td><td>550589</td><td></td></tr><tr><td>Matrigel GFR</td><td>BD Bioscience</td><td>356231</td><td></td></tr><tr><td>Collagenase A</td><td>Roche</td><td>10103586001</td><td></td></tr><tr><td>Mouse anti P75</td><td>Chemicon</td><td>MAB 365</td><td></td></tr><tr><td>11.0 Wire</td><td>Ethicon</td><td>FG 2881</td><td></td></tr><tr><td>Toluidine Blue&amp;nbsp;</td><td>Ral Diagnostics</td><td>361590-0025</td><td></td></tr><tr><td>Centrifuge</td><td>Sigma</td><td>Sigma 2-16PK</td><td></td></tr><tr><td>Incubator&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Laminar flow hood</td><td>Faster</td><td>BH-EN 2003 S</td><td></td></tr><tr><td>Flow cytometer</td><td>BD Bioscience</td><td>FACSCalibur</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Videolaryngoscope</td><td>Karl Storz Endoskope</td><td>Telecam SL NLSC 20212120</td><td></td></tr><tr><td>Acquisition system</td><td>AD Instruments</td><td>Powerlab system</td><td></td></tr><tr><td>Pyramitome Ultramicrotomy System&amp;nbsp;</td><td>Leica</td><td>Ultracut S</td><td></td></tr><tr><td>Image analysis system</td><td>Explora Nova</td><td>Mercator</td><td></td></tr></tbody></table>

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

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 shows ...

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 ...

The overall goal of this procedure is to present a model of cellular transplantation based on the use of olfactory and sheathing cells in a laryngeal nerve injury model. This is accomplished by first sectioning and performing anastomosis of the recurrent laryngeal nerve. Next, the olfactory and sheathing cells obtained from olfactory mucosa or olfactory bulbs are transplanted.Then two to three months after surgery, the animals are evaluated by laryngoscopy and electromyography. Finally, the recurrent laryngeal nerve is isolated for histological studies. Ultimately, results can be obtained that show olfactory and sheathing cells improving axonal regrowth of the recurrent laryngeal nerve and functional recovery of the vocal cords through laryngoscopy, electromyography, and histological measurements.This method can answer key questions in the field of peripheral nerve injury that involve the measure of axonal growth and the ability to quantify functionality of the recovery. Generally, individuals new to this method will struggle because recurrent lal nerves anastomosis require microsurgical skills After generating GFP labeled primary cultures of olfactory bulbs, or OB and olfactory mucosa or OM as outlined in the written protocol. Prepare for surgery by autoclaving all instruments.Once the rat has been anesthetized by intraperitoneal injection of ketamine and chlorpromazine, verify its level of sedation by performing a toe. Pinch, shave the surgical site and place the rat in dorsal decubitus, disinfect the skin with alternating washes of alcohol and Betadine solution. After that, assemble a sterile surgical environment and maintain sterile procedures throughout surgery.Use a scalpel to make a two centimeter medial cervical cutaneous incision. Next, following the medial white line, perform a vertical incision of the infra hyoid muscles using an orthostatic retractor between the infra hyoid muscles. Expose the larynx and then the recurrent laryngeal nerve or RLN.Then under a surgical microscope, use micro scissors to cut the nerve fully at the level of the seventh tracheal ring with one point of 11 T suture. Perform anastomosis to de innervate the larynx just before transplantation. Use 0.05%trypsin EDTA to remove the OB and OM cells from the dishes.Then to stop the digestion, add five milliliters of warm medium after centrifuging the cells at 432 G for five minutes. Use a hemo cytometer to count the cells and adjust the cellular concentration in this model between 1.2 and six times 10 to the six cells in 30 microliters of D-M-E-M-F 12 were used in a micro fuge tube. Prepare a one to two ratio of cells to growth factor, reduced matrigel and place on ice just prior to the transplantation.Remove the tube from the ice and hold it to warm the mixture. Using a pipette, depose the cell matrigel solution on the anastomosis site and allow the mixture to self-assemble on the injured nerve when it is set. Use four aught sutures to close the muscles and skin.Place the rat in an individual cage and keep it under a heating lamp for 24 hours. Administer analgesics for the next five days, approximately two months after surgery after anesthetizing. Rats as described earlier in this video, use a toe pinch to confirm sedation.Place the rat in dorsal decubitus before inserting a 30 degree video. Laryngoscope adjusted to provide the best view of the larynx to reproduce the same view For each recording. Determine anatomical landmarks such as the posterior commissure, the right vocal cord where it inserts into the vocal apophysis of the OID and the left focal cord where it inserts into the vocal hypothesis of the oid.Record vocal cord movements by selecting three sequences of successive maximal abduction and maximal abduction. To analyze laryngeal function, determine absolute angular movement by measuring the difference between the movement of maximal and maximal abduction of the vocal chords. Determine the dynamic score by measuring the movement amplitude and the functional score by measuring the synkinesis and paradoxical movements to carry out electromyography.Place an electrode on the hamstring muscle of the rat after exposing the larynx and trachea as demonstrated for recurrent nerve anastomosis. Expose the cricothyroid cartilage. Next, using micro scissors, open the cricothyroid cartilage to expose the posterior cricothyroid or PCA muscle.Insert a monopolar needle electrode into the muscle with the animal under spontaneous ventilation. Use an acquisition system to record the electrical muscular activity of the PCA muscle. Analyze the muscular activity in terms of richness and synchronization with the respiration by assigning a qualitative score from zero to three using the following scale.Zero equals an untried tracing without increase during inspiration. One equals a rhymed tracing with inspiration increasing, but poor tracing two equals a rhymed tracing with richer activity and three equals a rhymed tracing. That's very rich constituting an interference pattern similar to a maximal intentional activity.Next, to avoid damaging the RLN because of its small size, expose the vagus nerve, then attach an electrode and stimulate at this site. Record the latency and potential duration in the PCA muscle. Finally, after euthanizing the animal using a pentobarbital overdose under a microscope, locate the anastomosis, suture and remove the distal stump of the RLN between the anastomosis and larynx.Perform histological analysis according to the text protocol shown here after transplantation of cells from primary cultures of olfactory, mucosa and olfactory bulbs are examples of EMG traces obtained in the PCA muscle during respiration. Animals of the control group presented a rich electrical muscular activity with an increase during inspiration that was not present in the Rerated group. In this figure, histological analyses of the RLN by toluidine blue staining show typical fiber profiles from normal re innervated and transplanted animals.This measurement allows quantification of the number of myelinated fibers, the mean size of the fibers, and the mean thickness of the myelin as shown here. To complete the histological analyses. Tracking of GFP positive cells was performed after intra nervous transplantation in the sciatic nerve.This site was chosen rather than the RLN because the R lns small size prevents intra nervous transplantation. Hans master anastomosis of the recurrent perial nerve can be done in 15 minutes if it's performed properly After its development. These techniques paved the way for researcher in the field of peripheral nerve injury to explore the use of cellular transplantation in a rat model.After watching this video, you should have a good understanding how to transplant cells in the recurrent nerve injury injury model and evaluate functional recovery of the vocal cords through electromyography a lab endoscopy and histological measurements.

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Neuroscience

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