Name: coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration
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Dette arbeidet ble økonomisk støttet av prosjektet SAF2017-82736-C2-1-R fra Ministerio de Ciencia e Innovación til MTM-F og av Fundación Universidad Francisco de Vitoria til JS.

MERK: Dyreeksperimentering ble godkjent av nasjonale og institusjonelle bioetikkkomiteer.
1. ihOEG (Ts12 og Ts14) kultur
MERK: Denne prosedyren gjøres under sterile forhold i et biosikkerhetsskap for vevskultur.
<ol><li>Forbered 50 ml ME10 OEG kulturmedium som angitt i tabell 1.</li>
	<li>Forbered 5 ml DMEM/F12-FBS, som angitt i tabell 1, i et konisk rør på 15 ml.</li>
	<li>Varm begge medier ved 37 °C i et rent vannbad, i 15 m...

<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 transplantasjon på CNS skade steder regnes som en lovende terapi for CNS skade på grunn av sin utspekulerende pro-neuroregenerative egenskaper7,8,9. Men, avhengig av vevskilden- olfaktorisk slimhinne (OM-OEG) versus olfaktorisk pære (OB-OEG) - eller donorens alder, finnes det betydelig variasjon i en slik kapasitet26,31,33,<...

Voksne sentralnervesystemet (CNS) nevroner har begrenset regenerativ kapasitet etter skade eller sykdom. En vanlig strategi for å fremme CNS regenerering er transplantasjon, på skadestedet, av celletyper som induserer aksonal vekst som stamceller, Schwann-celler, astrocytter eller olfaktoriske ensheathing glia (OEG)celler 1,2,3,4,5.
OEG kommer fra nevrale crest6 og lokaliserer i olfaktorisk slimhinne og i olfactory pære. Hos voksne dør olfaktoriske sensoriske nevroner regelmessig som følge av miljøeksponering, og de erstattes av nylig differensierte nevroner. OEG omgir og guider disse nye olfaktoriske aksonene for å gå inn i olfaktorisk pære og å etablere nye synapser med sine mål i CNS7. På grunn av disse fysiologiske egenskapene har OEG blitt brukt i modeller av CNS-skade som ryggmarg eller optisk nerveskade og dens nevroregenerative og nevrobeskyttende egenskaper blir bevist8,9,10,11. Flere faktorer har blitt identifisert som ansvarlig for de pro-regenerative egenskapene til disse cellene, inkludert ekstracellulær matrise proteaser produksjon eller sekresjon av nevrotrofiske og aksonalevekstfaktorer 12,13,14.
Gitt de tekniske begrensningene for å utvide primære OEG-celler, har vi tidligere etablert og karakterisert reversibel udødeliggjort menneskelig OEG (ihOEG) kloniske linjer, som gir en ubegrenset tilførsel av homogen OEG. Disse ihOEG cellene stammer fra primære kulturer, utarbeidet av olfaktoriske pærer oppnådd i obduksjoner. De ble udødeliggjort ved transduksjon av telomerase katalytisk underenhet (TERT) og onkogen Bmi-1 og modifisert med SV40-viruset stort T antigen15,16,17,18. To av disse ihOEG cellelinjer er Ts14, som opprettholder regenerativ kapasitet av de opprinnelige kulturer og Ts12, en lav regenerativ linje som brukes som en lav regenereringskontroll i disse eksperimentene18.
For å vurdere OEG kapasitet til å fremme aksonær regenerering etter nevrale skader, har flere in vitro-modeller blitt implementert. I disse modellene brukes OEG på kulturer av forskjellig nevronal opprinnelse og neurittdannelse og forlengelse – som svar på glialkokultur – analyseres. Eksempler på slike nevronale kilder er neonatal rottekortikale nevroner19, scratch sår utført på rotte embryonale nevroner fra kortikaltvev 20, rotte retinal explants21, rotte hypothalamus eller hippocampal postnatal nevroner22,23, postnatal rotte dorsal rot ganglion nevroner24, postnatal mus kortikospinalkanal nevroner25, menneskelige NT2 nevroner26, eller postnatal cerebral kortikale nevroner på reaktiv astrocytt arr-lignende kulturer27.
I disse modellene er regenereringsanalysen imidlertid avhengig av embryonale eller postnatale nevroner, som har en iboende plastisitet som er fraværende i skadede voksne nevroner. For å overvinne denne ulempen, vi presentere en modell av voksen aksonal regenerering i cocultures av OEG linjer med voksen retinal ganglion nevroner (RGNs), basert på den som opprinnelig ble utviklet av Wigley et al.28,29,30,31 og modifisert og brukes av vår gruppe12,13,14,15,16,17,18,32,33. Kort, retinal vev ekstraheres fra voksne rotter og fordøyd med papain. Retinal celle suspensjon er deretter belagt på enten polylysin-behandlet coverslips eller på Ts14 og Ts12 monolayers. Kulturer opprettholdes i 96 timer før de er faste og deretter immunofluorescence for aksonære (MAP1B og NF-H proteiner)34 og somatodendritic (MAP2A og B)35 markører utføres. Aksonal regenerering kvantifiseres som en prosentandel av nevroner med akson, med hensyn til den totale populasjonen av RGN-er og aksonær regenereringsindeks beregnes som gjennomsnittlig aksonal lengde per nevron. Denne protokollen er ikke bare nyttig for å vurdere det neuroregenerative potensialet til ihOEG, men kan utvides til forskjellige kilder til OEG eller andre glialceller.

<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

Olfactory ensheathing glia (OEG) celler er lokalisert hele veien fra olfactory mucosa til og inn i olfactory nerve lag (ONL) av olfactory pære. Gjennom hele voksenlivet er de nøkkelen til aksonal dyrking av nygenererte olfaktoriske nevroner, fra lamina propria til ONL. På grunn av deres pro-regenerative egenskaper har disse cellene blitt brukt til å fremme aksonal regenerering i ryggmargen eller optiske nerveskademodeller.
Vi presenterer en in vitro modell for å analysere og måle OEG nevroregenerativ kapasitet etter nevrale skader. I denne modellen er reversibel udødeliggjort menneskelig OEG (ihOEG) dyrket som monolayer, netthinnen ekstraheres fra voksne rotter og retinal ganglion nevroner (RGN) er cocultured på OEG monolayer. Etter 96 timer analyseres aksonære og somatodendrittiske markører i RGN-er ved immunofluorescence og antall RGN-er med akson og gjennomsnittlig aksonal lengde/nevron kvantifiseres.
Denne protokollen har fordelen over andre in vitro-analyser som er avhengige av embryonale eller postnatale nevroner, at den evaluerer OEG neuroregenerative egenskaper i voksenvev. Det er heller ikke bare nyttig for å vurdere det neuroregenerative potensialet til ihOEG, men kan utvides til forskjellige kilder til OEG eller andre glialceller.

I denne protokollen presenterer vi en in vitro-modell for å analysere OEG nevroregenerativ kapasitet etter nevronal skade. Som vist i figur 1,OEG kilden er en reversibel udødeliggjort menneskelig OEG klonisk cellelinje -Ts14 og Ts12-, som stammer fra primære kulturer, utarbeidet av olfactory pærer oppnådd iobduksjoner 15,17,18. Retinal vev ekstraheres fra voksne rotter, fordøyd, og retinal gan...

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

Vi presenterer en in vitro modell for å vurdere olfactory ensheathing glia (OEG) neuroregenerative kapasitet, etter neural skade. Den er basert på en coculture av akotomiserte voksne retinal ganglion nevroner (RGN) på OEG monolayers og påfølgende studie av aksonal regenerering, ved å analysere RGN aksonale og somatodendritiske markører.

Coculture av akkotomiserte rotte Retinal Ganglion nevroner med Olfactory Ensheathing Glia, som en In Vitro modell av voksen aksonal regenerering

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

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

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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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Genetisk studie av Axon Regeneration med dyrkede Voksen røttene 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 ...

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Transplantasjon av Olfactory Ensheathing Cells å evaluere Funksjonell Recovery etter perifer nerveskade

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

En<em&gt; In-vitro</em&gt; Utarbeidelse av Isolerte Enteriske nevroner og gliaceller fra myenteric Plexus av Adult Mouse

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

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Retrograd Merking av retinal ganglion celler i voksen sebrafisk med fluorescerende fargestoffer

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

Luktsystemet som en modell for å studere aksonal vekst mønstre og morfologi<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

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Den Indirekte Neuron-astrocyte coculture analyse: En<em&gt; In Vitro</em&gt; Oppsett for detaljert undersøkelse av Neuron-gliaceller interaksjoner

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

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Müller Glia celle aktivering i en Laser-indusert Retinal degenerasjon og Regeneration modell i sebrafisk

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

Undersøker pattedyr Axon gjenfødelse: I Vivo Electroporation av voksen mus 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 ...