Name: coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration
Uploaded: 2020-11-02T15:00:00
Description: 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

Dit werk werd financieel ondersteund door project SAF2017-82736-C2-1-R van Ministerio de Ciencia e Innovación tot MTM-F en door Fundación Universidad Francisco de Vitoria aan JS.

OPMERKING: Dierproeven werden goedgekeurd door nationale en institutionele bio-ethiekcomités.
1. ihOEG (Ts12 en Ts14) cultuur
OPMERKING: Deze procedure wordt uitgevoerd onder steriele omstandigheden in een bioveiligheidskast voor weefselkweek.
<ol><li>Bereid 50 ml ME10 OEG-kweekmedium voor zoals vermeld in tabel 1.</li>
	<li>Bereid 5 ml DMEM/F12-FBS, zoals vermeld in tabel 1,in een conische buis van 15 ml.</li>
	<li>Verwarm beide...

<ol>
	<li>Kanno, H., Pearse, D. D., Ozawa, H., Itoi, E., Bunge, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+transplantation+for+spinal+cord+injury+repair:+Its+significant+therapeutic+potential+and+prospectus.">Schwann cell transplantation for spinal cord injury repair: Its significant therapeutic potential and prospectus.</a> Reviews in the Neurosciences. 26 (2), 121-128 (2015).</li><li>Assinck, P., Duncan, G. J., Hilton, B. J., Plemel, J. R., Tetzlaff, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+transplantation+therapy+for+spinal+cord+injury.">Cell transplantation therapy for spinal cord injury.</a> Nature Neuroscience. 20 (5), 637-647 (2017).</li><li>Lindsay, S. L., Toft, A., Griffin, J., Emraja, A. M. M., Barnett, S. C., Riddell, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+olfactory+mesenchymal+stromal+cell+transplants+promote+remyelination+and+earlier+improvement+in+gait+coordination+after+spinal+cord+injury.">Human olfactory mesenchymal stromal cell transplants promote remyelination and earlier improvement in gait coordination after spinal cord injury.</a> Glia. 65 (4), 639-656 (2017).</li><li>Moreno-Flores, M. T., 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=A+clonal+cell+line+from+immortalized+olfactory+ensheathing+glia+promotes+functional+recovery+in+the+injured+spinal+cord.">A clonal cell line from immortalized olfactory ensheathing glia promotes functional recovery in the injured spinal cord.</a> Molecular Therapy. 13 (3), 598-608 (2006).</li><li>Gilmour, A. D., Reshamwala, R., Wright, A. A., 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=Optimizing+olfactory+ensheathing+cell+transplantation+for+spinal+cord+injury+repair.">Optimizing olfactory ensheathing cell transplantation for spinal cord injury repair.</a> Journal of Neurotrauma. 37 (5), 817-829 (2020).</li><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> Proceedings of the National Academy of Sciences of the United States of America. 107, 21040-21045 (2010).</li><li>Su, Z., He, 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:+biology+in+neural+development+and+regeneration.">Olfactory ensheathing cells: biology in neural development and regeneration.</a> Progress in Neurobiology. 92 (4), 517-532 (2010).</li><li>Yao, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+for+spinal+cord+injury:+sniffing+out+the+issues.">Olfactory ensheathing cells for spinal cord injury: sniffing out the issues.</a> Cell Transplant. 27 (6), 879-889 (2018).</li><li>G&#243;mez, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy+for+spinal+cord+injury+with+olfactory+ensheathing+glia+cells+(OECs).">Cell therapy for spinal cord injury with olfactory ensheathing glia cells (OECs).</a> Glia. 66 (7), 1267-1301 (2018).</li><li>Plant, G. W., Harvey, A. R., Leaver, S. G., Lee, S. V. <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:+repairing+injury+to+the+mammalian+visual+system.">Olfactory ensheathing glia: repairing injury to the mammalian visual system.</a> Experimental Neurology. 229 (1), 99-108 (2011).</li><li>Xue, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplanted+olfactory+ensheathing+cells+restore+retinal+function+in+a+rat+model+of+light-induced+retinal+damage+by+inhibiting+oxidative+stress.">Transplanted olfactory ensheathing cells restore retinal function in a rat model of light-induced retinal damage by inhibiting oxidative stress.</a> Oncotarget. 8 (54), 93087-93102 (2017).</li><li>Pastrana, E., 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=Genes+associated+with+adult+axon+regeneration+promoted+by+olfactory+ensheathing+cells:+a+new+role+for+matrix+metalloproteinase+2.">Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2.</a> The Journal of Neuroscience. 26, 5347-5359 (2006).</li><li>Pastrana, E., 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=BDNF+production+by+olfactory+ensheathing+cells+contributes+to+axonal+regeneration+of+cultured+adult+CNS+neurons.">BDNF production by olfactory ensheathing cells contributes to axonal regeneration of cultured adult CNS neurons.</a> Neurochemistry International. 50, 491-498 (2007).</li><li>Sim&#243;n, 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=Expression+of+plasminogen+activator+inhibitor-1+by+olfactory+ensheathing+glia+promotes+axonal+regeneration.">Expression of plasminogen activator inhibitor-1 by olfactory ensheathing glia promotes axonal regeneration.</a> Glia. 59, 1458-1471 (2011).</li><li>Lim, F., 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=Reversibly+immortalized+human+olfactory+ensheathing+glia+from+an+elderly+donor+maintain+neuroregenerative+capacity.">Reversibly immortalized human olfactory ensheathing glia from an elderly donor maintain neuroregenerative capacity.</a> Glia. 58, 546-558 (2010).</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=Prevention+of+senescence+progression+in+reversibly+immortalized+human+ensheathing+glia+permits+their+survival+after+deimmortalization.">Prevention of senescence progression in reversibly immortalized human ensheathing glia permits their survival after deimmortalization.</a> Molecular Therapy. 18, 394-403 (2010).</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=A+neuroregenerative+human+ensheathing+glia+cell+line+with+conditional+rapid+growth.">A neuroregenerative human ensheathing glia cell line with conditional rapid growth.</a> Cell Transplant. 20, 153-166 (2011).</li><li>Plaza, N., Sim&#243;n, D., Sierra, J., Moreno-Flores, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+an+immortalized+olfactory+ensheathing+glia+cell+line+with+the+green+fluorescent+protein+(GFP)+gene:+Evaluation+of+its+neuroregenerative+capacity+as+a+proof+of+concept.">Transduction of an immortalized olfactory ensheathing glia cell line with the green fluorescent protein (GFP) gene: Evaluation of its neuroregenerative capacity as a proof of concept.</a> Neuroscience Letters. 612, 25-31 (2016).</li><li>Deumens, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alignment+of+glial+cells+stimulates+directional+neurite+growth+of+CNS+neurons+in+vitro.">Alignment of glial cells stimulates directional neurite growth of CNS neurons in vitro.</a> Neuroscience. 125 (3), 591-604 (2004).</li><li>Chung, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+promote+neurite+sprouting+of+injured+axons+in+vitro+by+direct+cellular+contact+and+secretion+of+soluble+factors.">Olfactory ensheathing cells promote neurite sprouting of injured axons in vitro by direct cellular contact and secretion of soluble factors.</a> Cell and Molecular Life Sciences. 61 (10), 1238-1245 (2004).</li><li>Leaver, S. G., Harvey, A. R., Plant, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+olfactory+ensheathing+glia+promote+the+long-distance+growth+of+adult+retinal+ganglion+cell+neurites+in+vitro.">Adult olfactory ensheathing glia promote the long-distance growth of adult retinal ganglion cell neurites in vitro.</a> Glia. 53 (5), 467-476 (2006).</li><li>Pellitteri, R., Spatuzza, M., Russo, A., Stanzani, S. <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+exert+a+trophic+effect+on+the+hypothalamic+neurons+in+vitro.">Olfactory ensheathing cells exert a trophic effect on the hypothalamic neurons in vitro.</a> Neuroscience Letters. 417 (1), 24-29 (2007).</li><li>Pellitteri, R., Spatuzza, M., Russo, A., Zaccheo, D., Stanzani, S. <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+represent+an+optimal+substrate+for+hippocampal+neurons:+an+in+vitro+study.">Olfactory ensheathing cells represent an optimal substrate for hippocampal neurons: an in vitro study.</a> International Journal of Developmental Neuroscience. 27 (5), 453-458 (2009).</li><li>Runyan, S. A., Phelps, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+olfactory+ensheathing+glia+enhance+axon+outgrowth+on+a+myelin+substrate+in+vitro.">Mouse olfactory ensheathing glia enhance axon outgrowth on a myelin substrate in vitro.</a> Experimental Neurology. 216 (1), 95-104 (2009).</li><li>Witheford, M., Westendorf, K., Roskams, A. 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+cells+promote+corticospinal+axonal+outgrowth+by+a+L1+CAM-dependent+mechanism.">Olfactory ensheathing cells promote corticospinal axonal outgrowth by a L1 CAM-dependent mechanism.</a> Glia. 61 (11), 1873-1889 (2013).</li><li>Roloff, F., Ziege, S., Baumg&#228;rtner, W., Wewetzer, K., Bicker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell-free+adult+canine+olfactory+ensheathing+cell+preparations+from+olfactory+bulb+and+mucosa+display+differential+migratory+and+neurite+growth-promoting+properties+in+vitro.">Schwann cell-free adult canine olfactory ensheathing cell preparations from olfactory bulb and mucosa display differential migratory and neurite growth-promoting properties in vitro.</a> BMC Neuroscience. 14, 141 (2013).</li><li>Khankan, R. R., Wanner, I. B., Phelps, P. E. <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+cell-neurite+alignment+enhances+neurite+outgrowth+in+scar-like+cultures.">Olfactory ensheathing cell-neurite alignment enhances neurite outgrowth in scar-like cultures.</a> Experimental Neurology. 269, 93-101 (2015).</li><li>Wigley, C. B., Berry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+adult+rat+retinal+ganglion+cell+processes+in+monolayer+culture:+comparisons+between+cultures+of+adult+and+neonatal+neurons.">Regeneration of adult rat retinal ganglion cell processes in monolayer culture: comparisons between cultures of adult and neonatal neurons.</a> Brain Research. 470 (1), 85-98 (1988).</li><li>Sonigra, R. J., Brighton, P. C., Jacoby, J., Hall, S., 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=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>

OEG-transplantatie op CZS-letselplaatsen wordt beschouwd als een veelbelovende therapie voor CZS-letsel vanwege de constitutieve pro-neuroregeneratieve eigenschappen7,8,9. Afhankelijk van de weefselbron — reukslijmvlies (OM-OEG) versus reukbol (OB-OEG)— of de leeftijd van de donor bestaat er echter aanzienlijke variatie in dergelijke capaciteit26,31,<sup c...

Volwassen centrale zenuwstelsel (CZS) neuronen hebben beperkte regeneratieve capaciteit na letsel of ziekte. Een gemeenschappelijke strategie ter bevordering van CZS-regeneratie is transplantatie, op de plaats van verwonding, van celtypen die axonale groei induceren, zoals stamcellen, Schwann-cellen, astrocyten of reukverwarmende gliacellen (OEG)1,2,3,4,5.
OEG is afgeleid van de neurale top6 en bevindt zich in het reukslijmvlies en in de reukbol. Bij volwassenen sterven reuksensorische neuronen regelmatig als gevolg van blootstelling aan het milieu en worden ze vervangen door nieuw gedifferentieerde neuronen. OEG omringt en begeleidt deze nieuwe reuk-axonen om de reukbol in te voeren en nieuwe synapsen met hun doelen in de CNS7vast te stellen. Vanwege deze fysiologische eigenschappen is OEG gebruikt in modellen van CZS-letsel zoals ruggenmerg- of oogzenuwletsel en zijn neuroregeneratieve en neuroprotectieve eigenschappen worden bewezen8,9,10,11. Verschillende factoren zijn geïdentificeerd als verantwoordelijk voor de pro-regeneratieve kenmerken van deze cellen, waaronder extracellulaire matrix proteasenproductie of afscheiding van neurotrofe en axonale groeifactoren12,13,14.
Gezien de technische beperkingen om primaire OEG-cellen uit te breiden, hebben we eerder omkeerbare vereeuwigde menselijke OEG (ihOEG) clonale lijnen opgericht en gekenmerkt, die een onbeperkte toevoer van homogene OEG bieden. Deze ihOEG-cellen zijn afkomstig van primaire culturen, bereid uit reukbollen verkregen in autopsies. Ze werden vereeuwigd door transductie van de telomerase katalytische subeenheid (TERT) en de oncogene Bmi-1 en gewijzigd met het SV40-virus groot T-antigeen15,16,17,18. Twee van deze ihOEG-cellijnen zijn Ts14, die de regeneratieve capaciteit van de oorspronkelijke culturen behoudt en Ts12, een lage regeneratieve lijn die wordt gebruikt als een lage regeneratiecontrole in deze experimenten18.
Om het OEG-vermogen te beoordelen om axonale regeneratie na neurale verwonding te bevorderen, zijn verschillende in vitro modellen geïmplementeerd. In deze modellen wordt OEG toegepast op culturen van verschillende neuronale oorsprong en worden neurietvorming en rek - als reactie op glia-cocultuur - getest. Voorbeelden van dergelijke neuronale bronnen zijn neonatale rat corticale neuronen19, kraswonden uitgevoerd op rat embryonale neuronen uit corticale weefsel20, rat retinale explants21, rat hypothalamus of hippocampale postnatale neuronen22,23, postnatale rat dorsale wortel ganglion neuronen24,postnatale muis corticospinale tractus neuronen25, menselijke NT2 neuronen26, of postnatale cerebrale corticale neuronen op reactieve astrocyte litteken-achtige culturen27.
In deze modellen is de regeneratietest echter gebaseerd op embryonale of postnatale neuronen, die een intrinsieke plasticiteit hebben die afwezig is bij gewonde volwassen neuronen. Om dit nadeel te overwinnen, presenteren we een model van volwassen axonale regeneratie in coculturen van OEG-lijnen met volwassen retinale ganglionneuronen (RGNs), gebaseerd op het model oorspronkelijk ontwikkeld door Wigley et al.28,29,30,31 en gewijzigd en gebruikt door onze groep12,13,14,15,16,17,18,32,33. Kortom, netvliesweefsel wordt geëxtraheerd uit volwassen ratten en verteerd met papaïne. Retinale celsuspensie wordt vervolgens geplateerd op met polylysine behandelde coverslips of op Ts14- en Ts12-monolagen. Culturen worden gedurende 96 uur bewaard voordat ze worden gefixeerd en vervolgens wordt immunofluorescentie uitgevoerd voor axonale (MAP1B- en NF-H-eiwitten)34 en somatodendritische (MAP2A en B)35 markers. Axonale regeneratie wordt gekwantificeerd als een percentage neuronen met axon, met betrekking tot de totale populatie RGN's en de axonale regeneratie-index wordt berekend als de gemiddelde axonale lengte per neuron. Dit protocol is niet alleen nuttig voor het beoordelen van het neuroregeneratieve potentieel van ihOEG, maar kan worden uitgebreid naar verschillende bronnen van OEG of andere gliacellen.

<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

Olfactorische ensheathing glia (OEG) cellen zijn gelokaliseerd helemaal van het reukslijmvlies tot en in de reukzenuwlaag (ONL) van de reukbol. Gedurende het hele volwassen leven zijn ze de sleutel tot de axonale groei van nieuw gegenereerde reukneuronen, van de lamina propria tot het ONL. Vanwege hun pro-regeneratieve eigenschappen zijn deze cellen gebruikt om axonale regeneratie in ruggenmerg- of oogzenuwletselmodellen te bevorderen.
We presenteren een in vitro model om OEG neuroregeneratieve capaciteit na neurale verwonding te testen en te meten. In dit model wordt reversibel vereeuwigd menselijk OEG (ihOEG) gekweekt als een monolaag, netvliezen worden geëxtraheerd uit volwassen ratten en retinale ganglionneuronen (RGN) worden samengecultiveerd op de OEG-monolaag. Na 96 uur worden axonale en somatodendritische markers in RGN's geanalyseerd door immunofluorescentie en wordt het aantal RGN's met axon en de gemiddelde axonlengte/neuron gekwantificeerd.
Dit protocol heeft het voordeel ten opzichte van andere in vitro tests die afhankelijk zijn van embryonale of postnatale neuronen, dat het OEG neuroregeneratieve eigenschappen in volwassen weefsel evalueert. Het is ook niet alleen nuttig voor het beoordelen van het neuroregeneratieve potentieel van ihOEG, maar kan worden uitgebreid naar verschillende bronnen van OEG of andere gliacellen.

In dit protocol presenteren we een in vitro model om het neuroregeneratieve vermogen van OEG na neuronaal letsel te testen. Zoals getoond in figuur 1, is de OEG-bron een omkeerbare vereeuwigde menselijke OEG clonale cellijn -Ts14 en Ts12-, die afkomstig is uit primaire culturen, bereid uit reukbollen verkregen in autopsies15,17,18. Retinale weefsel wordt geëxtraheerd uit volwassen ratten, verteerd,...

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

We presenteren een in vitro model om olfactorisch verwarmend glia (OEG) neuroregeneratief vermogen te beoordelen, na neuraal letsel. Het is gebaseerd op een cocultuur van axotomized volwassen retinale ganglion neuronen (RGN) op OEG monolagen en daaropvolgende studie van axonale regeneratie, door het analyseren van RGN axonale en somatodendritische markers.

Cocultuur van Axotomized Rat Retinal Ganglion Neurons met Olfactory Ensheathing Glia, als een In Vitro Model van Volwassen Axonale Regeneratie

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 ...

Research

JoVE Journal

Neuroscience

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfectie van Mouse retinale ganglioncellen door In vivo Elektroporatie

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

Bilaminar Co-cultuur van primaire Rat corticale neuronen en 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 ...

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

Genetische Studie van de regeneratie van axonen met gekweekte volwassen dorsale wortel ganglion neuronen

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

Transplantatie van olfactorische Ensheathing Cellen tot functioneel herstel Evalueer na perifere zenuwschade

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

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

Een<em&gt; In-vitro</em&gt; Voorbereiding van Geïsoleerde enterische neuronen en Glia uit de myenterische Plexus van de Adult Mouse

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

Retrograde Labeling van retinale ganglioncellen in Adult zebravis met fluorescente kleurstoffen

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

Het olfactorisch systeem als model om axonale groeipatronen en morfologie Bestudeer<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

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

De indirecte Neuron-astrocyten Coculture Assay: An<em&gt; In Vitro</em&gt; Set-up voor het gedetailleerd onderzoek van het neuron-glia interacties

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

MÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¼ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

Müller Glia cel activatie in een Laser-geïnduceerde Retina degeneratie en regeneratie Model in de zebravis

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

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

Behandelende zoogdieren Axon regeneratie: In Vivo Electroporation van volwassen muis achterwortelganglia 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 ...