与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型
Summary
我们提出了一个体外模型，以评估神经损伤后嗅觉包裹胶质（OEG）神经再生能力。它基于OEG单层的轴突成人视网膜结节神经元（RGN）的共生和随后的轴再生研究，通过分析RGN轴突和声乐内皮标记。
Abstract
嗅觉包裹胶质 （OEG） 细胞从嗅觉粘膜一直定位到嗅球的嗅觉神经层 （ONL）。在整个成年生活中，它们是新产生的嗅觉神经元的轴突生长的关键，从拉米纳预言家到ONL。由于其亲再生特性，这些细胞已被用于促进脊髓或视神经损伤模型的轴突再生。
我们提出了一个体外模型，以检测和测量神经损伤后OEG神经再生能力。在这个模型中，可逆不朽的人类OEG（ihOEG）被培养为单层，视网膜从成年大鼠中提取，视网膜结石神经元（RGN）被共同培养到OEG单层。96小时后，通过免疫荧光分析RGN中的轴突和索马托内皮标记，并量化带轴突的RN数量和平均轴突长度/神经元。
此协议比依赖胚胎或产后神经元的其他体外检测具有优势，它评估成人组织中的OEG神经再生特性。此外，它不仅可用于评估 ihOEG 的神经再生潜力，而且可以扩展到 OEG 或其他胶质细胞的不同来源。
Introduction
成人中枢神经系统（CNS）神经元在受伤或患病后再生能力有限。促进CNS再生的一个共同策略是移植，在损伤部位，诱导轴突生长的细胞类型，如干细胞，施万细胞，星形细胞或嗅觉包裹胶质（OEG）细胞1，2，3，4，5。
OEG源自神经峰6，位于嗅觉粘膜和嗅球中。在成人中，嗅觉神经元由于环境暴露而定期死亡，并被新分化的神经元所取代。OEG包围和引导这些新的嗅觉轴突进入嗅球，并建立新的突触，他们的目标在CNS7。由于这些生理属性，OEG已被用于CNS损伤的模型，如脊髓或视神经损伤，其神经再生和神经保护特性被证实为8，9，10，11。几个因素已被确定为负责这些细胞的亲再生特征，包括细胞外基质蛋白酶生产或分泌神经营养和轴突生长因子12，13，14。
鉴于扩大原OEG细胞的技术局限性，我们以前建立并定性了可逆不朽的人类OEG（ihOEG）克隆系，它提供了无限的均匀OEG供应。这些 ihOEG 细胞源自原始培养物，这些培养物来自验尸中获得的嗅觉球茎。它们通过端粒酶催化亚单（TERT）和上基因Bmi-1的转导而永生，并与SV40病毒大T抗原15、16、17、18一起改良。其中两个ihOEG细胞系是Ts14，它保持了原始培养物和Ts12的再生能力，Ts12是一种低再生线，在这些实验中用作低再生控制。
为了评估OEG在神经损伤后促进轴突再生的能力，已经实施了几个体外模型。在这些模型中，OEG应用于不同神经元起源和中性形成和拉长的培养物——对胶质文化的反应——被检测。这些神经元来源的例子有新生儿大鼠皮质神经元19，从皮质组织对大鼠胚胎神经元进行划伤20，大鼠视网膜外泄21，大鼠下丘脑或海马产后神经元22，23 ，产后大鼠鼻脊髓根结节神经元24个，产后小鼠皮质脊髓神经元25个，人类NT2神经元26个，或产后脑皮质神经元对反应性星形细胞疤痕样培养27个。
然而，在这些模型中，再生检测依赖于胚胎或产后神经元，这些神经元具有受伤的成人神经元所缺乏的内在可塑性。为了克服这个缺点， 我们提出了一个成人轴突再生模型在OEG线的培养与成人视网膜结节神经元（RN），基于一个最初由威格利等人28，29，30，31和修改和使用我们组12，13，14，15，16，17，18，32，33。简言之，视网膜组织从成年大鼠中提取，用木瓜消化。视网膜细胞悬浮液随后被镀在多晶硅处理的盖片上或 Ts14 和 Ts12 单层上。培养物在修复前保持96小时，然后进行轴突（MAP1B和NF-H蛋白）34和体血体（MAP2A和B）35标记的免疫荧光。轴突再生被量化为轴突神经元的百分比，关于RGN的总数，轴再生指数被计算为每个神经元的平均轴突长度。此协议不仅可用于评估 ihOEG 的神经再生潜力，而且可以扩展到 OEG 或其他胶质细胞的不同来源。
Protocol
注：动物实验已获国家和机构生物伦理委员会批准。 1. ihOEG （Ts12 和 Ts14） 文化 注：此程序是在组织培养生物安全柜的无菌条件下完成的。 准备 表 1中提供的 50 mL ME10 OEG 文化介质。 准备5毫升的DMEM/F12-FBS，如 表1所示，在15毫升圆锥管。 在37°C的清洁水浴中加热两种介质15分钟。 在干净的水浴中，在…
Representative Results
在此协议中，我们提出了一个体外模型，以检测神经元损伤后的OEG神经再生能力。如图1所示，OEG源是一个可逆的不朽的人类OEG克隆细胞系-Ts14和Ts12-，它源自原始文化，从验尸15，17，18获得的嗅球。视网膜组织从成年大鼠身上提取，消化后，视网膜结节神经元（RGN）悬浮物镀在PLL处理的盖片上或iHOEG单层?…
Discussion
OEG移植在CNS损伤部位被认为是一个有希望的治疗CNS损伤，因为它构成亲神经再生属性7，8，9。然而，根据组织来源-嗅觉粘膜（OM-OEG）与嗅球（OB-OEG）-或捐赠者的年龄，这种能力存在相当大的差异26，31，33，36。因此，在开始体内?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了SAF2017-82736-C2-1-R项目的财政支持，该项目由西恩西亚·埃因诺瓦西翁部长到MTM-F，由弗朗西斯科·德维托里亚基金会向JS提供。
Materials
| antibody 514 | Reference 34 | Rabbit polyclonal antiserum, which recognizes MAP2A and B. | |
| antibody SMI-31 | BioLegend | 801601 | Monoclonal antibody against MAP1B and NF-H proteins |
| anti-mouse Alexa Fluor 488 antibody | ThermoFisher | A-21202 | |
| anti-rabbit Alexa Fluor 594 antibody | ThermoFisher | A-21207 | |
| B-27 Supplement | Gibco | 17504044 | |
| D,L-2-amino-5-phosphonovaleric acid | Sigma | 283967 | NMDA receptor inhibitor |
| DAPI | Sigma | D9542 | Nuclei fluorescent stain |
| DMEM-F12 | Gibco | 11320033 | Cell culture medium |
| FBS | Gibco | 11573397 | Fetal bovine serum |
| FBS-Hyclone | Fisher Scientific | 16291082 | Fetal bovine serum |
| Fluoromount | Southern Biotech | 0100-01 | Mounting medium |
| ImageJ | National Institutes of Health (NIH-USA) | Image software | |
| L-Glutamine | Lonza | BE17-605F | |
| Neurobasal Medium | Gibco | 21103049 | Neuronal cells culture medium |
| Papain Dissociation System | Worthington Biochemical Corporation | LK003150 | For use in neural cell isolation |
| PBS | Home made | ||
| PBS-EDTA | Lonza | H3BE02-017F | |
| Penicillin/Streptomycin/Amphotericin B | Lonza | 17-745E | Bacteriostatic and bactericidal |
| Pituitary extract | Gibco | 13028014 | Bovine pituitary extract |
| Poly -L- lysine (PLL) | Sigma | A-003-M |
Riferimenti
- Kanno, H., Pearse, D. D., Ozawa, H., Itoi, E., Bunge, M. B. Schwann cell transplantation for spinal cord injury repair: Its significant therapeutic potential and prospectus. Reviews in the Neurosciences. 26 (2), 121-128 (2015).
- Assinck, P., Duncan, G. J., Hilton, B. J., Plemel, J. R., Tetzlaff, W. Cell transplantation therapy for spinal cord injury. Nature Neuroscience. 20 (5), 637-647 (2017).
- Lindsay, S. L., Toft, A., Griffin, J., Emraja, A. M. M., Barnett, S. C., Riddell, J. S. Human olfactory mesenchymal stromal cell transplants promote remyelination and earlier improvement in gait coordination after spinal cord injury. Glia. 65 (4), 639-656 (2017).
- Moreno-Flores, M. T., et al. A clonal cell line from immortalized olfactory ensheathing glia promotes functional recovery in the injured spinal cord. Molecular Therapy. 13 (3), 598-608 (2006).
- Gilmour, A. D., Reshamwala, R., Wright, A. A., Ekberg, J. A. K., St. John, J. A. Optimizing olfactory ensheathing cell transplantation for spinal cord injury repair. Journal of Neurotrauma. 37 (5), 817-829 (2020).
- Barraud, P., et al. Neural crest origin of olfactory ensheathing glia. Proceedings of the National Academy of Sciences of the United States of America. 107, 21040-21045 (2010).
- Su, Z., He, C. Olfactory ensheathing cells: biology in neural development and regeneration. Progress in Neurobiology. 92 (4), 517-532 (2010).
- Yao, R., et al. Olfactory ensheathing cells for spinal cord injury: sniffing out the issues. Cell Transplant. 27 (6), 879-889 (2018).
- Gómez, R. M., et al. Cell therapy for spinal cord injury with olfactory ensheathing glia cells (OECs). Glia. 66 (7), 1267-1301 (2018).
- Plant, G. W., Harvey, A. R., Leaver, S. G., Lee, S. V. Olfactory ensheathing glia: repairing injury to the mammalian visual system. Experimental Neurology. 229 (1), 99-108 (2011).
- Xue, L., et al. Transplanted olfactory ensheathing cells restore retinal function in a rat model of light-induced retinal damage by inhibiting oxidative stress. Oncotarget. 8 (54), 93087-93102 (2017).
- Pastrana, E., et al. Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2. The Journal of Neuroscience. 26, 5347-5359 (2006).
- Pastrana, E., et al. BDNF production by olfactory ensheathing cells contributes to axonal regeneration of cultured adult CNS neurons. Neurochemistry International. 50, 491-498 (2007).
- Simón, D., et al. Expression of plasminogen activator inhibitor-1 by olfactory ensheathing glia promotes axonal regeneration. Glia. 59, 1458-1471 (2011).
- Lim, F., et al. Reversibly immortalized human olfactory ensheathing glia from an elderly donor maintain neuroregenerative capacity. Glia. 58, 546-558 (2010).
- García-Escudero, V., et al. Prevention of senescence progression in reversibly immortalized human ensheathing glia permits their survival after deimmortalization. Molecular Therapy. 18, 394-403 (2010).
- García-Escudero, V., et al. A neuroregenerative human ensheathing glia cell line with conditional rapid growth. Cell Transplant. 20, 153-166 (2011).
- Plaza, N., Simón, D., Sierra, J., Moreno-Flores, M. T. 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. Neuroscience Letters. 612, 25-31 (2016).
- Deumens, R., et al. Alignment of glial cells stimulates directional neurite growth of CNS neurons in vitro. Neuroscienze. 125 (3), 591-604 (2004).
- Chung, R. S., et al. Olfactory ensheathing cells promote neurite sprouting of injured axons in vitro by direct cellular contact and secretion of soluble factors. Cell and Molecular Life Sciences. 61 (10), 1238-1245 (2004).
- Leaver, S. G., Harvey, A. R., Plant, G. W. Adult olfactory ensheathing glia promote the long-distance growth of adult retinal ganglion cell neurites in vitro. Glia. 53 (5), 467-476 (2006).
- Pellitteri, R., Spatuzza, M., Russo, A., Stanzani, S. Olfactory ensheathing cells exert a trophic effect on the hypothalamic neurons in vitro. Neuroscience Letters. 417 (1), 24-29 (2007).
- Pellitteri, R., Spatuzza, M., Russo, A., Zaccheo, D., Stanzani, S. Olfactory ensheathing cells represent an optimal substrate for hippocampal neurons: an in vitro study. International Journal of Developmental Neuroscience. 27 (5), 453-458 (2009).
- Runyan, S. A., Phelps, P. E. Mouse olfactory ensheathing glia enhance axon outgrowth on a myelin substrate in vitro. Experimental Neurology. 216 (1), 95-104 (2009).
- Witheford, M., Westendorf, K., Roskams, A. J. Olfactory ensheathing cells promote corticospinal axonal outgrowth by a L1 CAM-dependent mechanism. Glia. 61 (11), 1873-1889 (2013).
- Roloff, F., Ziege, S., Baumgärtner, W., Wewetzer, K., Bicker, G. Schwann cell-free adult canine olfactory ensheathing cell preparations from olfactory bulb and mucosa display differential migratory and neurite growth-promoting properties in vitro. BMC Neuroscience. 14, 141 (2013).
- Khankan, R. R., Wanner, I. B., Phelps, P. E. Olfactory ensheathing cell-neurite alignment enhances neurite outgrowth in scar-like cultures. Experimental Neurology. 269, 93-101 (2015).
- Wigley, C. B., Berry, M. Regeneration of adult rat retinal ganglion cell processes in monolayer culture: comparisons between cultures of adult and neonatal neurons. Brain Research. 470 (1), 85-98 (1988).
- Sonigra, R. J., Brighton, P. C., Jacoby, J., Hall, S., Wigley, C. B. Adult rat olfactory nerve ensheathing cells are effective promoters of adult central nervous system neurite outgrowth in coculture. Glia. 25 (3), 256-269 (1999).
- Hayat, S., Thomas, A., Afshar, F., Sonigra, R., Wigley, C. B. Manipulation of olfactory ensheathing cell signaling mechanisms: effects on their support for neurite regrowth from adult CNS neurons in coculture. Glia. 44 (3), 232-241 (2003).
- Kumar, R., Hayat, S., Felts, P., Bunting, S., Wigley, C. Functional differences and interactions between phenotypic subpopulations of olfactory ensheathing cells in promoting CNS axonal regeneration. Glia. 50 (1), 12-20 (2005).
- Moreno-Flores, M. T., Lim, F., Martín-Bermejo, M. J., Díaz-Nido, J., Avila, J., Wandosell, F. Immortalized olfactory ensheathing glia promote axonal regeneration of rat retinal ganglion neurons. Journal of Neurochemistry. 85 (4), 861-871 (2003).
- García-Escudero, V., et al. Patient-derived olfactory mucosa cells but not lung or skin fibroblasts mediate axonal regeneration of retinal ganglion neurons. Neuroscience Letters. 509 (1), 27-32 (2012).
- Sternberger, L. A., Sternberger, N. H. Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proceedings of the National Academy of Sciences of the United States of America. 80 (19), 6126-6130 (1983).
- Sánchez Martin, C., Díaz-Nido, J., Avila, J. Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons. Neuroscienze. 87 (4), 861-870 (1998).
- Reshamwala, R., Shah, M., Belt, L., Ekberg, J. A. K., St. John, J. A. Reliable cell purification and determination of cell purity: crucial aspects of olfactory ensheathing cell transplantation for spinal cord repair. Neural Regeneration Research. 15 (11), 2016-2026 (2020).
Citazione di questo articolo
Portela-Lomba, M., Simón, D., Russo, C., Sierra, J., Moreno-Flores, M. T. Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration. J. Vis. Exp. (165), e61863, doi:10.3791/61863 (2020).