Ces travaux ont été soutenus financièrement par le projet SAF2017-82736-C2-1-R de Ministerio de Ciencia e Innovación à MTM-F et par fundación Universidad Francisco de Vitoria à JS.

REMARQUE : L’expérimentation animale a été approuvée par les comités nationaux et institutionnels de bioéthique.
1. culture ihOEG (Ts12 et Ts14)
REMARQUE : Cette procédure se fait dans des conditions stériles dans une armoire de biosécurité de culture tissulaire.
<ol><li>Préparez 50 mL me10 milieu de culture OEG tel que fourni dans le tableau 1.</li>
	<li>Préparer 5 mL de DMEM/F12-FBS, tel que prévu dans le tableau 1,dans un tube conique de 15 mL.</li>
	<li>Réchauffer les deux supports à 37 °C dans un bain d’eau propre, pendant 15 min.</li>
	<li>Décongeler les flacons des cellules Ts12 et Ts14 à 37 °C dans un bain d’eau propre.</li>
	<li>Resuspendez et ajoutez des cellules au milieu de culture DMEM/F12-FBS préparé à l’étape 2.</li>
	<li>Centrifugeuse pendant 5 min à 200 x g.</li>
	<li>Aspirez au surnatant.</li>
	<li>Ajouter 500 μL de ME10 moyen et réutiliser la pastille.</li>
	<li>Préparer un plat de culture cellulaire p60 avec 3 mL de ME10 et ajouter la suspension cellulaire, dropwise.</li>
	<li>Déplacez-vous pour répartir les cellules uniformément sur la plaque.</li>
	<li>Cellules de culture à 37 °C dans 5% DE CO2. REMARQUE : Après avoir atteint la confluence, au moins un autre passage doit être fait pour optimiser les cellules pour la coculture. 90% de confluence est nécessaire avant de les ensemencer sur les coverslips pour la coculture. Un confluent p-60 a un nombre moyen de cellules de 7 x 105 pour Ts14 et 2,5 x 106 pour les lignées cellulaires Ts12. Les lignées cellulaires Ts12 et Ts14 doivent être passages tous les 2 à 3 jours.</li>
</ol>2. Préparation de l’ihOEG (Ts12 et Ts14) pour l’essai
REMARQUE : Cette étape doit être effectuée 24 h avant la dissection et la coculture du RGN.
<ol><li>Traiter les couvre-lips Ø de 12 mm avec 10 μg/mL poly-L-lysine (PLL) pendant 1 h. REMARQUE : Les coverslips peuvent être laissés pendant la nuit dans la solution PLL.</li>
	<li>Lavez les coverslips avec 1x phosphate tampon salin (PBS), trois fois.</li>
	<li>Détachez les cellules Ts12 et Ts14 ihOEG du plat de culture cellulaire p60.<ol>
<li>Ajouter 4 mL de milieu de culture DMEM/F12-FBS(tableau 1)à un tube conique de 15 mL. Chaud à 37 °C dans un bain d’eau propre.</li>
		<li>Retirer le milieu des assiettes et laver les cellules avec 1 mL de 1x PBS-EDTA, une fois.</li>
		<li>Ajouter 1 mL de trypsine-EDTA aux cellules OEG et incuber pendant 3-5 min à 37 °C, 5 % de CO2.
</li>
		<li>Recueillir les cellules avec une pipette p1000 et les transférer à moyen préparé à l’étape 3.1.</li>
		<li>Centrifugeuse pendant 5 min à 200 x g.</li>
		<li>Aspirez au surnatant.</li>
		<li>Ajouter 1 mL de ME10 moyen et réutiliser la pastille.</li>
		<li>Comptez le numéro de cellule dans un hémocytomètre.</li>
	</ol>
</li>
	<li>Ensemencer 80 000 cellules Ts14 ou 100 000 cellules Ts12/puits sur les couvertures dans des plaques de 24 puits dans 500 μL de me10 moyen.</li>
	<li>Cellules de culture à 37 °C à 5 % de CO2, pendant 24 h.</li>
</ol>3. Dissection des tissus rétiniens
REMARQUE : Les rats Wistar mâles de 2 mois sont utilisés comme source RGN. Deux rétines (un rat) pour 20 puits d’un plat cellulaire de 24 puits. Autoclave matériel chirurgical avant utilisation. Papain kit de dissociation est acheté commercialement (Table of Materials). Suivez les instructions du fournisseur pour la reconstitution. Reconstituer D, L-2-amino-5-phosphonovaleric acide (APV) dans le stock de 5 mM et préparer les aliquots.
<ol><li>Le jour de l’analyse, préparez les médias suivants.<ol>
<li>Préparer un plat de culture cellulaire p60 avec 5 mL d’EBSS froid (flacon 1 du kit de dissociation de la papaïne).</li>
		<li>Préparer un plat de culture cellulaire p60 avec du flacon reconstitué 2 (papain) du kit de dissociation de la papaïne plus 50 μL d’APV.  Ensuite, ajoutez 250 μL de flacon reconstitué 3 (DNase plus 5 μL d’APV).</li>
		<li>Dans un tube stérile, mélanger 2,7 mL de flacon 1 avec 300 μL de flacon 4 (inhibiteur de la protéase albumin-ovomucoïde). Ajouter 150 μL de flacon 3 (DNase) plus 30 μL d’APV.</li>
		<li>Préparer 20 mL de milieu Neurobasal-B27 (NB-B27) tel que fourni dans le tableau 1.</li>
	</ol>
</li>
	<li>Sacrifier un rat par asphyxie avec du CO2.</li>
	<li>Retirer la tête par décapitation avec de la guillotine; placez-le dans une boîte de Pétri de 100 mm et vaporisez la tête d’éthanol à 70 % avant de la placer dans un capot d’écoulement laminaire.</li>
	<li>Couper les moustaches du rat avec des ciseaux afin qu’ils n’interfèrent pas avec la manipulation des yeux.</li>
	<li>Saisir le nerf optique avec des forceps pour retirer le globe oculaire assez pour être en mesure de faire une incision à travers l’œil avec un scalpel.</li>
	<li>Retirez la lentille et l’humour vitreux et retirez la rétine (tissu orange), tandis que les couches restantes de l’œil restent à l’intérieur (y compris la couche épithéliale pigmentaire).</li>
	<li>Placez la rétine dans le plat de culture cellulaire p60 préparé à l’étape 3.1.1.</li>
	<li>Transférer la rétine dans le plat de culture cellulaire p60 préparé à l’étape 3.1.2 et le couper avec le scalpel en petits morceaux d’une taille approximative < 1 mm.</li>
	<li>Transférer dans un tube en plastique de 15 mL.</li>
	<li>Incuber le tissu pendant 30 min, dans un incubateur humidifié à 37 °C sous 5% de CO2,avec agitation toutes les 10 minutes.</li>
	<li>Dissocier les touffes de cellules en pipetage de haut en bas avec une pipette pasteur en verre.</li>
	<li>Centrifugeuse de la suspension cellulaire à 200 x g pendant 5 min.</li>
	<li>Jetez le supernatant et inactivez la papaïne, réutilisez la pastille cellulaire dans la solution préparée à l’étape 3.1.3. (1,5 mL pour 2 yeux).</li>
	<li>Pipet soigneusement cette suspension cellulaire en 5 mL de flacon reconstitué 4.</li>
	<li>Centrifugeuse à 200 x g pendant 5 min.</li>
	<li>Pendant la centrifugeuse, retirez complètement le milieu ME-10 de la plaque cellulaire du puits OEG 24 (préalablement préparée à l’étape 2) et remplacez-la par 500 μL de NB-B27 moyen par puits.</li>
	<li>Jetez le supernatant et resuspendez les cellules dans 2 mL de milieu NB-B27.</li>
	<li>Plaque 100 μL de suspension cellulaire rétinienne, par puits de la plaque m24, sur des monolayers-coverslips PLL ou OEG.</li>
	<li>Maintenir les cultures à 37 °C avec 5 % de CO2 pendant 96 h dans le milieu du NB-B27.</li>
</ol>4. Immunostaining
<ol><li>Après 96 h, fixer les cellules pendant 10 min en ajoutant le même volume de 4% de paraformaldéhyde (PFA) en 1x PBS au milieu de culture (600 μL) (concentration finale PFA 2%).</li>
	<li>Retirez le média et le PFA de la plaque 24-multiwell et ajoutez encore une fois 500 μL de 4% de paraformaldéhyde (PFA) en 1x PBS. Incuber pendant 10 min.</li>
	<li>Jeter le fixateur et laver 3 fois avec 1x PBS pendant 5 min.</li>
	<li>Bloc avec 0,1% Triton X-100/1% FBS en PBS (PBS-TS) pendant 30-40 min.</li>
	<li>Préparez les anticorps primaires dans le tampon PBS-TS comme suit : SMI31 (contre les protéines MAP1B et NF-H) anticorps monoclonaux (1:500). 514 (reconnaît les protéines MAP2A et B) antisérum polyclonal de lapin (1:400).</li>
	<li>Ajouter des anticorps primaires aux cocultures et incuber toute la nuit à 4 °C.</li>
	<li>Le lendemain, jetez les anticorps et lavez les coverslips avec 1x PBS, 3 fois, pendant 5 min.</li>
	<li>Préparez les anticorps secondaires dans le tampon PBS-TS comme suit: Pour SMI-31, anti-souris Alexa Fluor 488 (1:500). Pour 514, anti-lapin Alexa-594 (1:500).</li>
	<li>Incuber les cellules avec les anticorps secondaires fluorescents correspondants pendant 1 h, à RT, dans l’obscurité.</li>
	<li>Laver les coverslips avec 1x PBS, 3 fois, pendant 5 min, dans l’obscurité.</li>
	<li>Enfin, montez des coverslips avec support de montage(Table of Materials)et maintenez-les à 4 °C. REMARQUE : Chaque fois que nécessaire, des noyaux fluorescents souillant avec DAPI (4,6-diamidino-2-phenylindole) peuvent être exécutés. Avant de monter, incuber les cellules pendant 10 min dans l’obscurité avec dapi (10 μg/mL en 1x PBS). Lavez les coverslips 3 fois avec 1x PBS et enfin, montez les coverslips avec le support de montage.</li>
</ol>5. Quantification de régénération axonale
REMARQUE : Les échantillons sont quantifiés dans le cadre de l’objectif 40x d’un microscope à épifluorescence. Un minimum de 30 photos doivent être prises sur des champs aléatoires, avec au moins 200 neurones, à quantifier pour chaque traitement. Chaque expérience doit être répétée au moins trois fois.
<ol><li>Quantifier le pourcentage de neurones avec l’axone (SMI31 neurite positif) par rapport à la population totale de RGNs (identifié avec MAP2A/B 514 immunostaining positif du corps neuronal et des dendrites).</li>
	<li>Quantifier l’indice de régénération axonale ou la longueur axonale moyenne (μm/neurone). Ce paramètre est défini comme la somme des longueurs (en μm) de tous les axones identifiés, divisés par le nombre total de neurones comptés, qu’ils présentaient ou non un axone. La longueur axonale est déterminée à l’aide du plugin NeuronJ du logiciel d’image ImageJ (NIH-USA).</li>
	<li>Calculez la signification moyenne, l’écart type et la signification statistique à l’aide du logiciel approprié.</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+olfactory+nerve+ensheathing+cells+are+effective+promoters+of+adult+central+nervous+system+neurite+outgrowth+in+coculture.">Adult rat olfactory nerve ensheathing cells are effective promoters of adult central nervous system neurite outgrowth in coculture.</a> Glia. 25 (3), 256-269 (1999).</li><li>Hayat, S., Thomas, A., Afshar, F., Sonigra, R., Wigley, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+olfactory+ensheathing+cell+signaling+mechanisms:+effects+on+their+support+for+neurite+regrowth+from+adult+CNS+neurons+in+coculture.">Manipulation of olfactory ensheathing cell signaling mechanisms: effects on their support for neurite regrowth from adult CNS neurons in coculture.</a> Glia. 44 (3), 232-241 (2003).</li><li>Kumar, R., Hayat, S., Felts, P., Bunting, S., Wigley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differences+and+interactions+between+phenotypic+subpopulations+of+olfactory+ensheathing+cells+in+promoting+CNS+axonal+regeneration.">Functional differences and interactions between phenotypic subpopulations of olfactory ensheathing cells in promoting CNS axonal regeneration.</a> Glia. 50 (1), 12-20 (2005).</li><li>Moreno-Flores, M. T., Lim, F., Mart&#237;n-Bermejo, M. J., D&#237;az-Nido, J., Avila, J., Wandosell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+olfactory+ensheathing+glia+promote+axonal+regeneration+of+rat+retinal+ganglion+neurons.">Immortalized olfactory ensheathing glia promote axonal regeneration of rat retinal ganglion neurons.</a> Journal of Neurochemistry. 85 (4), 861-871 (2003).</li><li>Garc&#237;a-Escudero, V., 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=Patient-derived+olfactory+mucosa+cells+but+not+lung+or+skin+fibroblasts+mediate+axonal+regeneration+of+retinal+ganglion+neurons.">Patient-derived olfactory mucosa cells but not lung or skin fibroblasts mediate axonal regeneration of retinal ganglion neurons.</a> Neuroscience Letters. 509 (1), 27-32 (2012).</li><li>Sternberger, L. A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proceedings of the National Academy of Sciences of the United States of America. 80 (19), 6126-6130 (1983).</li><li>S&#225;nchez Martin, C., D&#237;az-Nido, J., Avila, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+a+site-specific+phosphorylation+of+the+microtubule-associated+protein+2+during+the+development+of+cultured+neurons.">Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons.</a> Neuroscience. 87 (4), 861-870 (1998).</li><li>Reshamwala, R., Shah, M., Belt, L., Ekberg, J. A. K., St. John, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+cell+purification+and+determination+of+cell+purity:+crucial+aspects+of+olfactory+ensheathing+cell+transplantation+for+spinal+cord+repair.">Reliable cell purification and determination of cell purity: crucial aspects of olfactory ensheathing cell transplantation for spinal cord repair.</a> Neural Regeneration Research. 15 (11), 2016-2026 (2020).</li></ol>

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La transplantation d’OEG aux emplacements de dommages de CNS est considérée une thérapie prometteuse pour des dommages de CNS dus à ses propriétés pro-neurorégénérativesconstitutives 7,8,9. Toutefois, selon la source tissulaire — muqueuse olfactive (OM-OEG) par rapport à l’ampoule olfactive (OB-OEG) — ou l’âge du donneur, il existe des variations considérables dans cettecapacité 26<...

Les neurones du système nerveux central adulte (SNC) ont une capacité de régénération limitée après une blessure ou une maladie. Une stratégie commune pour favoriser la régénération de CNS est la transplantation, au site de blessure, des types de cellules qui induisent la croissance axonale telle que des cellules souches, des cellules de Schwann, des astrocytes ou des cellules olfactives d’ensheathing glia (OEG)1,2,3,4,5.
OEG dérive de la crêteneurale 6 et se trouve dans la muqueuse olfactive et dans le bulbe olfactif. Chez l’adulte, les neurones sensoriels olfactifs meurent régulièrement à la suite de l’exposition à l’environnement et ils sont remplacés par des neurones nouvellement différenciés. OEG entoure et guide ces nouveaux axones olfactifs pour entrer dans l’ampoule olfactive et établir de nouvelles synapses avec leurs cibles dans le CNS7. En raison de ces attributs physiologiques, OEG a été employé dans des modèles des dommages de CNS tels que la moelle épinière ou la lésion de nerf optique et ses propriétés neuroregénératives et neuroprotective deviennentprouvées 8,9,10,11. Plusieurs facteurs ont été identifiés comme responsables des caractéristiques pro-régénératrices de ces cellules, y compris la production extracellulaire de protéases de matrice ou la sécrétion des facteurs neurotrophiques et axonalsde croissance 12,13,14.
Compte tenu des limitations techniques pour étendre les cellules OEG primaires, nous avons précédemment établi et caractérisé réversible immortalisé oeg humain (ihOEG) lignes clonales, qui fournissent un approvisionnement illimité d’OEG homogène. Ces cellules ihOEG proviennent de cultures primaires, préparées à partir d’ampoules olfactives obtenues lors d’autopsies. Ils ont été immortalisés par transduction de la sous-unité catalytique de télomérase (TERT) et de l’oncogène Bmi-1 et modifiés avec le virus SV40 grand antigène T15,16,17,18. Deux de ces lignées cellulaires ihOEG sont Ts14, qui maintient la capacité régénératrice des cultures originales et Ts12, une ligne régénérative faible qui est utilisée comme un faible contrôle de régénération dans ces expériences18.
Pour évaluer la capacité d’OEG à favoriser la régénération axonale après des dommages neuronaux, plusieurs modèles in vitro ont été mis en œuvre. Dans ces modèles, l’OEG est appliqué aux cultures d’origine neuronale différente et la formation et l’allongement de neurite- en réponse à la coculture gliale- sont assayed. Des exemples de telles sources neuronales sont les neurones corticals néonatals de rat19,les blessures d’éraflure exécutées sur les neurones embryonnaires de rat du tissu cortical20,les explants rétiniens de rat21,les neurones postnatals hypothalamiques ou hippocampal de rat22,23, neurones postnatals de ganglion de racine dorsale de rat24,neurones corticospinal postnatals de région de souris25,neurones humains de NT226,ou neurones corticals cérébraux postnatals sur les cultures réactives de cicatrice d’astrocyte27.
Dans ces modèles, cependant, l’analyse de régénération repose sur des neurones embryonnaires ou postnatals, qui ont une plasticité intrinsèque qui est absente dans les neurones adultes blessés. Pour surmonter cet inconvénient, nous présentons un modèle de régénération axonale adulte dans les cocultures de lignées OEG avec des neurones ganglionaux rétiniens adultes (RRG), basé sur celui développé à l’origine par Wigley et coll.28,29,30,31 et modifié et utilisé par notre groupe12,13,14,15,16,17,18,32,33. En bref, le tissu rétinien est extrait de rats adultes et digéré avec de la papaïne. La suspension cellulaire rétinienne est ensuite plaquée sur des couvre-plaques traités à la polylysine ou sur des monocouches Ts14 et Ts12. Les cultures sont maintenues pendant 96 h avant d’être fixées, puis l’immunofluorescence pour les protéines axonales (protéines MAP1B et NF-H)34 et les marqueurs somatodendritiques (MAP2A et B)35 sont effectués. La régénération axonale est quantifiée en pourcentage des neurones avec l’axone, en ce qui concerne la population totale de RRG et l’indice de régénération axonale est calculée comme la longueur axonale moyenne par neurone. Ce protocole est non seulement utile pour évaluer le potentiel neurorégénératif de l’ihOEG, mais peut être étendu à différentes sources d’OEG ou d’autres cellules gliales.

<table>
	<tbody>
		<tr>
			<td>antibody 514</td>
			<td>Reference 34</td>
			<td></td>
			<td>Rabbit polyclonal antiserum, which recognizes MAP2A and B.</td>
		</tr>
		<tr>
			<td>antibody SMI-31</td>
			<td>BioLegend</td>
			<td>801601</td>
			<td>Monoclonal antibody against MAP1B and NF-H proteins</td>
		</tr>
		<tr>
			<td>anti-mouse Alexa Fluor 488 antibody</td>
			<td>ThermoFisher</td>
			<td>A-21202</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa Fluor 594 antibody</td>
			<td>ThermoFisher</td>
			<td>A-21207</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>D,L-2-amino-5-phosphonovaleric acid</td>
			<td>Sigma</td>
			<td>283967</td>
			<td>NMDA receptor inhibitor</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Nuclei fluorescent stain</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>11573397</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>FBS-Hyclone</td>
			<td>Fisher Scientific</td>
			<td>16291082</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Fluoromount</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health (NIH-USA)</td>
			<td></td>
			<td>Image software</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605F</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Neuronal cells culture medium</td>
		</tr>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td>For use in neural cell isolation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-EDTA</td>
			<td>Lonza</td>
			<td>H3BE02-017F</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Amphotericin B</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>Bacteriostatic and bactericidal</td>
		</tr>
		<tr>
			<td>Pituitary extract</td>
			<td>Gibco</td>
			<td>13028014</td>
			<td>Bovine pituitary extract</td>
		</tr>
		<tr>
			<td>Poly -L- lysine (PLL)</td>
			<td>Sigma</td>
			<td>A-003-M</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration

Les cellules olfactives d’ensheathing glia (OEG) sont localisées tout le chemin de la muqueuse olfactive à et dans la couche olfactive de nerf (ONL) de l’ampoule olfactive. Tout au long de la vie adulte, ils sont essentiels à la croissance axonale des neurones olfactifs nouvellement générés, de la propria lamina à l’ONL. En raison de leurs propriétés pro-régénératives, ces cellules ont été utilisées pour favoriser la régénération axonale dans les modèles de lésions de la moelle épinière ou du nerf optique.
Nous présentons un modèle in vitro pour apaiser et mesurer la capacité neurorégénérative d’OEG après des dommages neuronaux. Dans ce modèle, l’OEG humain réversiblement immortalisé (ihOEG) est cultivé comme monocouche, les rétines sont extraites des rats adultes et les neurones rétiniens de ganglion (RGN) sont coculturels sur le monocouche d’OEG. Après 96 h, les marqueurs axonals et somatodendritiques dans les RGN sont analysés par immunofluorescence et le nombre de RGNs avec l’axone et la longueur/neurone axonal moyen sont quantifiés.
Ce protocole a l’avantage sur d’autres analyses in vitro qui reposent sur des neurones embryonnaires ou postnatals, qu’il évalue les propriétés neurorégénératives oeg dans les tissus adultes. En outre, il est non seulement utile pour évaluer le potentiel neurorégénératif de l’ihOEG, mais peut être étendu à différentes sources d’OEG ou d’autres cellules gliales.

Dans ce protocole, nous présentons un modèle in vitro pour apaiser la capacité neurorégénérative d’OEG après des dommages neuronaux. Comme le montre la figure 1, la source de l’OEG est une lignée cellulaire clonale humaine immortalisée réversible -Ts14 et Ts12-, qui provient de cultures primaires, préparées à partir d’ampoules olfactives obtenues dans les autopsies15,17,18. Le tis...

Watch this Scientific Journal Video about Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration at JoVE.com

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

Nous présentons un modèle in vitro pour évaluer la capacité neurorégénérative olfactive d’ensheathing de glia (OEG), après dommages neuronaux. Il est basé sur une coculture de neurones ganglionnaires rétiniens adultes axotomisés (RGN) sur les monocouches OEG et l’étude subséquente de la régénération axonale, en analysant les marqueurs axonals et somatodendritiques RGN.

Coculture de neurones rétiniens axotomisés de ganglion de rat avec glia ensheathing olfactif, comme modèle in vitro de régénération axonale adulte

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 ...

coculture-axotomized-rat-retinal-ganglion-neurons-with-olfactory

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JoVE Journal

Neuroscience

3.8K vues.  Universidad Francisco de Vitoria. Nous présentons un modèle in vitro pour évaluer la capacité neurorégénérative olfactive d’ensheathing de glia (OEG), après dommages neuronaux. Il est basé sur une coculture de neurones ganglionnaires rétiniens adultes axotomisés (RGN) sur les monocouches OEG et l’étude subséquente de la régénération axonale, en analysant les marqueurs axonals et somatodendritiques RGN.

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

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Didermique co-culture de neurones de rat corticale primaire et des cellules gliales

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

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Neurosciences

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Sélective transduction virale des adultes-né pour les neurones olfactifs chronique In vivo Stimulation optogénétique

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Étude génétique de la régénération des axones avec cultivées adultes dorsaux neurones ganglion de la racine

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

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.

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.

La transplantation de cellules olfactives engainantes à évaluer la récupération fonctionnelle après des nerfs périphériques blessures

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

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Une<em&gt; In-vitro</em&gt; Préparation des neurones entériques isolés et cellules gliales du Plexus myentérique de la souris adulte

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Marquage rétrograde des cellules ganglionnaires de la rétine à l&#39;âge adulte de poisson zèbre avec des colorants fluorescents

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

Le système olfactif comme un modèle pour étudier des modèles axonale croissance et la morphologie<em&gt; In Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Le Neuron-astrocyte indirecte coculture Assay: Un<em&gt; In vitro</em&gt; Mise en place pour l&#39;investigation détaillée des interactions Neuron-glie

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

M&amp;#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

Activation des cellules Müller Glia dans une dégénérescence rétinienne induite par Laser et le modèle de régénération chez le poisson zèbre

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Chargée de l’enquête la régénération axonale mammifères : In Vivo électroporation du Ganglion de la racine dorsale de souris adulte

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...