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

이 작품은 프로젝트 SAF2017-82736-C2-1-R 장관 드 시엔시아 e 이노바시온에서 MTM-F, Fundación Universidad Francisco de Vitoria에서 JS에 의해 재정적으로 지원되었다.

참고: 동물 실험은 국가 및 기관 생물 윤리 위원회에 의해 승인되었습니다.
1. ihOEG (Ts12 및 Ts14) 문화
참고: 이 절차는 조직 배양 생물 안전 캐비닛의 멸균 조건하에서 수행됩니다.
<ol><li>표 1에제공된 50mL ME10 OEG 배양 배지를 준비한다.</li>
	<li>표 1에제공된 대로 DMEM/F12-FBS 5mL을 15mL 원문 튜브에 준비합니다.</li>
	<li>깨끗한 수조에서 3...

<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>

CNS 상해 부위에서 OEG 이식은 구성적인 프로 신경 재생특성7,8,9로인해 CNS 부상에 대한 유망한 치료법으로 간주됩니다. 그러나, 조직원에 따라 후각점막(OM-OEG)과 후각전(OB-OEG)-또는 기증자의 나이에 따라, 이러한 용량26,31,33,36에상당...

성인 중추 신경계 (CNS) 뉴런은 부상이나 질병 후 재생 능력이 제한되어 있습니다. CNS 재생을 촉진하는 일반적인 전략은 줄기 세포, 슈완 세포, 성상세포 또는 후각 내성 신경교(OEG) 세포1,2,3,4,5와같은 축축성장을 유도하는 세포 유형의 부상부위에서이식된다.
OEG는 신경 문장6에서 파생되고 후각 점막과 후각 전구에서 찾습니다. 성인에서, 후각 감각 뉴런은 환경 노출의 결과로 정기적으로 죽고 새로 분화 된 뉴런으로 대체됩니다. OEG는 이러한 새로운 후각 축축을 둘러싸고 안내하여 후각 전구에 들어가 CNS7에서목표를 가진 새로운 시냅스를확립합니다. 이러한 생리적 특성으로 인해 OEG는 척수 나 시신경 손상과 같은 CNS 부상 의 모델에 사용되어 왔으며 신경 생성 및 신경 보호 특성은8,9,10,11로입증되었습니다. 세포외 매트릭스 프로테아제 생산 또는 신경영양 및 축삭 성장 인자12,13,14의분비를 포함한 이러한 세포의 재생 특성에 대한 여러 가지 요인으로 확인되었습니다.
1차 OEG 세포를 확장하는 기술적 한계를 감안할 때, 우리는 이전에 균일 한 OEG의 무제한 공급을 제공하는 가역 불멸의 인간 OEG (ihOEG) 클론 라인을 설립하고 특성화했습니다. 이러한 ihOEG 세포는 부검에서 얻은 후각 전구에서 제조된 1차 배양에서 유래한다. 그(것)들은 텔로머라아제 촉매 서브유닛 (TERT)과 종양균 Bmi-1의 전도에 의해 불멸되고 SV40 바이러스 큰 T 항원15,16,17,18로변형되었다. 이들 ihOEG 세포주 중 2개는 Ts14로, 이는 본래 배양과 Ts12의 재생 능력을 유지하며, 이러한 실험에서 낮은 재생 제어로 사용되는 낮은 재생성라인(18)이다.
신경 손상 후 축 하 재생을 육성 하는 OEG 용량을 평가 하기 위해, 여러 시험 관 모델이 구현 되었습니다. 이러한 모델에서, OEG는 서로 다른 뉴런 기원과 중성염 형성 및 신장의 배양에 적용되며, 이는 글리아교 공동 문화에 대한 반응으로 분석된다. 이러한 뉴런 소스의 예로는 신생아 쥐 피질뉴런(19)이있으며, 피질조직(20)에서쥐 배아 뉴런에 수행된 스크래치 상처, 쥐 망막 이병21,쥐 시상하또는 해마 산후 뉴런22,23,산후 쥐 등쪽 뿌리 신경절뉴런(24), 산후 마우스 코르티코피타피통뉴런(25,인간 NT2 뉴런26)또는 사후 뇌피질 뉴런이 반응성 성상세포 흉터와 같은 배양(27)을 한다.
그러나 이러한 모델에서는 재생 분석이 부상당한 성인 뉴런에 없는 본질적인 가소성을 가진 배아 또는 산후 뉴런에 의존합니다. 이러한 단점을 극복하기 위해, 우리는 원래 Wigley등28,29,30,31에 의해 개발된 것을 기반으로 성인 망막 신경절 뉴런(RGNs)과 OEG 라인의 공동 배양에서 성인 축축산 재생 모델을 제시하고 우리 그룹12,13,14, 15,15, 16,16, 17, 18, 18, 18, 18, 18, 17, 18, 18, 18, 18, 18, 18, 15, 16, 18,18, 18, 18, 18, 18, 18, 18, 18, 17, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 17, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 18, 17, 18, 18, 18, 18,1 간략하게, 망막 조직은 성인 쥐에서 추출하고 papain로 소화됩니다. 망막 세포 현탁액은 폴리리신 처리 커버립 또는 Ts14 및 Ts12 단층층상에 도금됩니다. 배양은 축산(MAP1B 및 NF-H 단백질)34 및 소마토덴드리틱(MAP2A 및 B)35마커를 고정하기 전에 96시간 동안 유지된다. 축사 재생은 축축을 가진 뉴런의 백분율로 정량화되며, RGNs및 축축 재생 지수의 총 인구에 대하여 뉴런당 평균 축축수 길이로 계산된다. 이 프로토콜은 ihOEG의 신경 재생 잠재력을 평가하는 데 유용할 뿐만 아니라 OEG 또는 다른 신경교 세포의 다른 소스로 확장 될 수 있습니다.

<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

후각 내시경 신경교 (OEG) 세포는 후각 점막에서 후각 전구의 후각 신경 층 (ONL)으로 모든 방법을 국소화된다. 성인 생활 내내, 그들은 새로 생성 된 후각 뉴런의 축 하 성장에 대 한 열쇠, 라미나 프로 프리 테리토리에서 ONL. 그들의 프로 재생 속성으로 인해, 이 세포는 척수 또는 시신경 상해 모형에 있는 축축재생을 육성하기 위하여 이용되었습니다.
우리는 신경 손상 후 OEG 신경 재생 용량을 분석하고 측정하기 위해 체외 모델을 제시합니다. 본 모델에서, 가역적으로 불멸의 인간 OEG(ihOEG)는 단층으로 배양되고, 망막은 성인 쥐로부터 추출되고 망막 신경절 뉴런(RGN)은 OEG 단층상에 공동 배양된다. 96h 후, RGNs의 축산 및 소종 절인 마커는 축축및 평균 축산 길이/뉴런을 가진 RG의 수에 의해 정량화된다.
이 프로토콜은 배아 또는 산후 뉴런에 의존하는 다른 체외 분석에 비해 장점이 있으며 성인 조직에서 OEG 신경 재생 특성을 평가합니다. 또한 ihOEG의 신경 재생 잠재력을 평가하는 데 유용할 뿐만 아니라 OEG 또는 다른 신경교 세포의 다른 소스로 확장 될 수 있습니다.

이 프로토콜에서, 우리는 신경 손상 후 OEG 신경 재생 용량을 분석하는 체외 모델을 제시한다. 도 1에도시된 바와 같이, OEG 소스는 부검15,17,18에서얻은 후각 전구로부터 제조된 1차 배양으로부터 유래한 가역불멸인간 OEG 클론 세포주-Ts12-이다. 망막 조직은 성인 쥐로부터 추출되고, 소화되고, 망막 신경절 ?...

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

우리는 신경 손상 후 후각 내성 glia (OEG) 신경 재생 능력을 평가하는 체외 모델을 제시한다. 그것은 RGN 축상 및 somatodritic 마커를 분석해서, OEG 단층 및 축 변성의 후속 연구 결과에 축축한 성인 망막 신경절 신경절 뉴런 (RGN)의 공동 문화를 기반으로합니다.

축 축 하 재생의 시험관 내 모델로, 후각 천시 글리아와 축 축 절 망막 신경의 공동 문화

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

마우스 망막 신경절 세포 Transfection로 생체내에 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 ...

연구

신경과학

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

차 쥐 두피 뉴런과 Glia의 Bilaminar의 공동 문화

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

교양 성인 지느러미 루트 신경절의 신경 세포와 축삭 재생의 유전 연구

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

말초 신경 손상 후 기능 회복을 평가하기 위해 후각 Ensheathing 세포의 이식

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

<em&gt; 체외</em성인 마우스 Myenteric 신경총에서 분리 된 장내 뉴런과 교세포의&gt; 준비

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

형광 염료와 성인 Zebrafish의 망막 신경절 세포의 퇴행성 라벨링

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

모델로 후각 시스템은 축삭 성장 패턴 및 형태를 연구하는<em&gt; 생체</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

간접 신경-성상 세포 공동 배양 분석 일 :<em&gt; 체외</em&gt; 신경 세포 - glia 상호 작용의 상세한 조사를위한 셋업

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

레이저 유도 된 망막 변성 및 재생 Zebrafish 모델 뮐러 명과 셀 활성화

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

성인 마우스 지 루트 신경 절의 Electroporation Vivo에서 조사 포유류 축 삭 재생:

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