小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测
Summary
在这里, 我们展示了一个最小的脊髓损伤模型的表现, 在一只成年小鼠, 备用的中心运河利基住房内源性神经干细胞。我们展示了如何使用干细胞检测来量化的确定性和原始神经干细胞的活化和迁移后损伤。
Abstract
成体哺乳动物脊髓中的神经干细胞是一种相对 mitotically 的静态脑室细胞, 可以用干细胞法进行体外研究。这种菌落形成法是研究神经干细胞对外源因子反应的有力工具;然而, 这也可以用来研究在体内操纵的效果, 正确理解的优势和局限性的检测。对临床感兴趣的一个操作是损伤对内源性神经干细胞活化的影响。目前的脊髓损伤模型为研究这一问题提供了一项挑战, 因为常见的挫伤、压迫和横断模型的严重性导致了干细胞驻留在损伤部位的国安区的破坏。在这里, 我们描述了一个最小的损伤模型, 造成局部损害在表面侧表面的下胸椎水平 (T7/8) 的成年小鼠脊髓。这种损伤模型在伤害的水平上, 将中央管放在损伤的高度, 并允许在损伤后的不同时间点上驻留在病变水平的神经干细胞进行分析。在这里, 我们展示如何利用干细胞检测来研究两个不同的, 直系相关的, 在脊髓脑室地区的神经干细胞 (pNSCs 和 dNSCs, 分别) 的种群的活化。我们展示了如何分离和培养这些神经干细胞从脑室地区的伤害水平和白质损伤部位。我们的术后脊髓解剖显示, 与对照组相比, 从受伤的脐带脑室区 pNSC 和 dNSC 衍生 neurospheres 的数量增加, 并通过损伤激活。此外, 在受伤后, dNSC 衍生的 neurospheres 可以从损伤部位分离出来–表明神经干细胞从脑室的位置迁移到受伤部位的能力。
Introduction
中枢神经系统包含自我更新, 多能干细胞的亚群, 有能力产生所有不同成熟的神经细胞类型1,2,3,4。这些神经干细胞位于大脑和脊髓的特殊位置, 在损伤后可以激活, 从而增殖、迁移和分化成成熟的神经细胞。神经干细胞及其子代已被证明迁移到损伤部位的皮质损伤模型5,6。在大脑中, 神经干细胞已被证明从侧脑室迁移到受伤部位, 在那里他们分化成星形胶质细胞, 导致胶质疤痕形成7。然而, 在脊髓中, 很少有研究来询问这些同种内源性神经干细胞能否被利用来促进脊髓损伤后的恢复。事实上, 目前有争论, 在脊髓干细胞池的活化是否需要直接物理损伤的脑室利基衬里的中央运河8或如果损伤脊髓实质 (离开茎细胞位完整) 足以激活内源性神经干细胞9。
许多脊髓损伤 (SCI) 模型被用来研究急性和慢性损伤的病理生理学。这些模型也被用来测试潜在的治疗方法治疗 SCI 通过神经保护, 免疫, 并发展细胞移植/置换策略10,11,13。目前的模型包括压缩和/或挫伤伤害, 造成大规模功能缺陷, 以及广泛的病变和空化在脐带14,15。由此产生的胶质疤痕可以跨越数个脊柱段和大部分的宽度/周长的脊髓16。因此, 尽管这些模型在临床上是相关的, 但它们对研究损伤后内源性神经干细胞的反应有很大的挑战。有化学模型的伤害, 可以适应造成更轻微的伤害形式, 可以腾出中央运河17。然而, 这些类型的伤害集中在与 sci 相关的脱髓鞘, 并没有临床相关模型的物理和/或机械损伤相关的创伤性 SCI。
为了解决目前的损伤模型的局限性, 我们已经适应了一个针轨最小的 SCI 模型, 最初开发的鼠9, 用于在成年鼠模型中的应用。我们的适应损伤模型可以造成小鼠脊髓侧区的一致病变, 并在损伤的水平上为中央管提供备用。这个模型的优点是它允许研究损伤后的神经干细胞动力学, 以及它们在损伤部位的潜在径向迁移。使用鼠标模型也允许使用转基因小鼠, 允许血统跟踪的内源性神经干细胞及其后代受伤后。神经干细胞的性质可以进一步评估使用的改良形式的体外干细胞检测, 这是在本议定书中介绍。
干细胞法是一种体外菌落形成试验, 允许在 mitogens 的存在下孤立神经干细胞。在克隆电镀密度, 个体神经干细胞增殖, 以产生自由浮动的球形细胞, 由神经干细胞的小亚群和绝大多数祖18,19组成。在我们的协议中, 我们展示了两个不同的, 直系相关的神经干细胞从脊髓脑室区的分离-在基线条件下, 并遵循我们最小的 SCI 模型。明确的神经干细胞 (dNSCs) 表达巢蛋白和胶质纤维酸性蛋白质 (GFAP) 和生长在存在的表皮生长因子 (EGF), 细胞生长因子, 和肝素 (一起称为经济适用房)20。这些 dNSCs 是罕见的在天真的脊髓, 导致很少 neurospheres的体外。然而, 我们表明, dNSCs 是激活后, 最小的 SCI, 扩大 neurospheres 的数量从脑室地区隔离21。原始神经干细胞 (pNSCs) 是 dNSCs 在神经干细胞谱系中的上游。pNSCs 是极其罕见的, 表达低水平的干细胞标记 Oct4, 和白血病抑制因子 (LiF) 反应22。pNSCs 在原代培养中由于髓鞘碱性蛋白 (MBP) 的存在而与成年小鼠脊髓隔绝, 不形成 neurospheres;然而, pNSC neurospheres 可以从蛋白缺乏小鼠分离出来, 他们的数量在伤害后扩大-类似于 dNSCs21。最后, 我们表明, dNSC 衍生 neurospheres 可以从早期的损伤部位与最小的 SCI 后分离。这些发现表明, 我们的损伤模型和分析可以评估脑室神经干细胞的活化特性, 如其增殖和迁移的能力, 以应对伤害。
Protocol
Representative Results
Discussion
在手术过程中, 有几个关键步骤, 研究者应该特别注意, 以获得最佳结果, 并尽量减少动物之间的变异性。在手术中必须注意吸入麻醉 (异氟醚), 因为麻醉已经被证明具有保护作用, 长期暴露27。因此, 在研究脊髓损伤后的再生能力时, 尽量快速有效地执行手术, 防止混淆变量。每只老鼠保持相同的异氟醚暴露时间将减少变异。在整个手术过程中, 老鼠的呼吸速度应该受到监控, 并且不?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由 Krembil 基金会 (运营赠款 CMM) 提供资金。”WX” 是玛格丽特？史密斯学生奖的接受者。他获得了安大略省的研究生奖学金。
Materials
| Agricola Retractor | Fine Science Tools | 17005-04 | |
| Moria Vannas-Wolff Spring Scissors (Curved) | Fine Science Tools | 15370-50 | Customize when ordering to get blunted tips |
| Graefe Forceps (Straight, 1×2 Teeth) | Fine Science Tools | 11053-10 | |
| Extra Fine Graefe Forceps (Curved, Serrated) | Fine Science Tools | 11152-10 | Or any other forceps for suturing |
| Hartman Hemostats (Straight) | Fine Science Tools | 13002-10 | Or any other appropriate for suturing |
| Scalpel Handle #3 | Fine Science Tools | 10003-12 | Or any other appropriate |
| Hair clippers | amazon.ca | https://www.amazon.ca/Wahl-Professional-8685-Classic-Clipper/dp/B00011K2BA | or any other appropriate |
| Stereotaxic instrument | Stoeling | 51500 | or any other appropriate |
| Buprenorphine | or any appropirate sanctioned my animal care facility | ||
| Meloxicam | or any appropriate sanctioned by animal care facility | ||
| Tears Naturale P.M. | Alcon | https://www.amazon.ca/Alcon-Tear-Gel-Liquid-Eye-Gel/dp/B00HHXGUXE | or any other appropriate |
| Isoflurane | Baxter International Inc | DIN 02225875 | or any other appropriate for anesthesia |
| Q-tips Cottom Swabs | amazon.ca | https://www.amazon.ca/Q-Tips-Cotton-Swabs-500-Count/dp/B003M5UO6U/ref=pd_lpo_vtph_194_bs_tr_img_1/140-7113119-8364127?_encoding=UTF8&psc=1&refRID=JC16N542KVRF2N62N3DS | |
| Cotton Gauze | Fisher Scientific | 13-761-52 | |
| 30G Needles | Becton Dickinson | 305106 | For Injury |
| 25G Needles | Becton Dickinson | 305122 | For Drug injections |
| 1mL Syringes | Becton Dickinson | 3090659 | for drug injections |
| 3mL Syringes | Becton Dickinson | 309657 | for fluid injections |
| 4-0 Suture | uoftmedstore.com | 2297-VS881 | for skin suturing |
| 6-0 Suture | uoftmedstore.com | VS889 | for muscle suturing |
| Polysporin ointment | amazon.ca | 102051 | |
| Isoflurane Vaporizer | VetEquip | 901806 | |
| 15mL conical tubes | ThermoFisher | Any appropriate | |
| Petri Dishes | ThermoFisher | any appropriate | |
| Trypan Blue | ThermoFisher | Any | |
| Hemocytometer | ThermoFisher | Any appropriate | |
| Centrifuge | ThermoFisher | Any appropriate | |
| Standard Dissection Tools | Fine Science Tools | ||
| Dissection Microscope | Zeiss | Stemi 2000 | |
| Counting Microscope | Olympus | CKX41 | |
| Neural Basal-A Medium | Invitrogen | 10888-022 | |
| B27 | Invitrogen | 17404-044 | |
| Penicillin- Streptomycin | Gibco | 15070 | |
| L- Glutamine | Gibco | 25030 | |
| DMEM | Invitrogen | 12100046 | |
| F12 | Invitrogen | 12700075 | |
| 30% Glucose | Sigma | G6152 | 1M- 9.01g in 100mL dH2O |
| 1M Glucose | |||
| 7.5% NaHCO3 | Sigma | S5761 | 155mM- 1.30g in 100mL dH2O |
| 155mM NaHCO3 | |||
| 1M HEPES | Sigma | H3375 | 23.83 g in 100mL dH2O |
| Apo-Transferrin | R&D Systems | 3188-AT | |
| Putrescine | Sigma | P7505 | |
| Insulin | Sigma | I5500 | |
| Selenium | Sigma | S9133 | |
| Progesterone | Sigma | P6149 | |
| Papain Dissociation System | Worthington Biochemical Corporation | PDS | 1 vial of papain can be used for 2 samples |
| Epidermal Growth Factor | Invitrogen | PMG8041 | Powder reconstituted with 1mL Hormone Mix and aliquoted into 20uL vials to be stored in freezer |
| Fibroblast Growth Factor | Invitrogen | PHG0226 | Powder reconstituted with 0.5mL Hormone Mix and aliquoted into 20uL vials to be stored in freezer |
| Heparin | Sigma | H3149 | |
| Leukemia Inhibitory Factor | In House | ||
| Trypan Blue | |||
| Hemocytometer | |||
| 24 well Plates | NUNC | ||
| 2M NaCl | Sigma | S5886 | 11.69g in 100mL dH2O |
| 1M KCL | Sigma | P5405 | 7.46g in 100mL dH2O |
| 1M MgCl2 | Sigma | M2393 | 20.33g in 100mL dH2O |
| 108mM CaCl2 | Sigma | C7902 | 1.59g in 100mL dH2O |
References
- Johansson, C. B., et al. Identification of a neural stem cell in the adult mammalian central nervous system. Cell. 96 (1), 25-34 (1999).
- McKay, R. Stem cells in the central nervous system. Science. 276 (5309), 66-71 (1997).
- Gage, F. H. Mammalian neural stem cells. Science. 287 (5457), 1433-1438 (2000).
- Temple, S., Alvarez-Buylla, A. Stem cells in the adult mammalian central nervous system. Current Opinion in Neurology. 9 (1), 135-141 (1999).
- Zhang, R., et al. Activated neural stem cells contribute to stroke-induced neurogenesis and neuroblast migration toward the infarct boundary in adult rats. Journal Of Cerebral Blood Flow And Metabolism. 24 (4), 441-448 (2004).
- Komitova, M., Mattsson, B., Johansson, B. B., Eriksson, P. S. Enriched environment increases neural stem/progenitor cell proliferation and neurogenesis in the subventricular zone of stroke-lesioned adult rats. Stroke. 36 (6), 1278-1282 (2005).
- Faiz, M., et al. Adult neural stem cells from the subventricular zone give rise to reactive astrocytes in the cortex after stroke. Cell Stem Cell. 17 (5), 624-634 (2015).
- Ren, Y., et al. Ependymal cell contribution to scar formation after spinal cord injury is minimal, local and dependent on direct ependymal injury. Science Reports – UK. 7, (2017).
- Mothe, A. J., Tator, C. H. Proliferation, migration, and differentiation of endogenous ependymal region stem/progenitor cells following minimal spinal cord injury in the adult rat. Neuroscience. 131 (1), 177-187 (2005).
- Thuret, S., Moon, L. D., Gage, F. H. Therapeutic interventions after spinal cord injury. Nature Reviews Neuroscience. 7 (8), 628-643 (2006).
- Bethea, J. R., et al. Systemically administered interleukin-10 reduces tumor necrosis factor-alpha production and significantly improves functional recovery following traumatic spinal cord injury in rats. Journal of Neurotrauma. 16 (10), 851-863 (1999).
- Donnelly, D. J., Popovich, P. G. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. Experimental Neurology. 209 (2), 378-388 (2008).
- Cummings, B. J., et al. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proceedings of the National Academy of Sciences USA. 102 (39), 14069-14074 (2005).
- Beattie, M. S., Hermann, G. E., Rogers, R. C., Bresnahan, J. C. Cell death in models of spinal cord injury. Progress in Brain Research. 137, 37-47 (2002).
- Metz, G. A., et al. Validation of the weight-drop contusion model in rats: a comparative study of human spinal cord injury. Journal of Neurotrauma. 17 (1), 1-17 (2000).
- Faulkner, J. R., et al. Reactive astrocytes protect tissue and preserve function after spinal cord injury. Journal of Neuroscience. 24 (9), 2143-2155 (2004).
- Cheriyan, T., et al. Spinal cord injury models: a review. Spinal Cord. 52 (8), 588-595 (2014).
- Deleyrolle, L. P., Reynolds, B. A. Isolation, expansion, and differentiation of adult Mammalian neural stem and progenitor cells using the neurosphere assay. Neural Cell Transplantation: Methods and Protocols. , 91-101 (2009).
- Singec, I., et al. Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nature Methods. 3 (10), (2006).
- Mignone, J. L., Kukekov, V., Chiang, A. S., Steindler, D., Enikolopov, G. Neural stem and progenitor cells in nestin-GFP transgenic mice. The Journal of Comparative Neurology. 469 (3), 311-324 (2004).
- Xu, W., et al. Myelin basic protein regulates primitive and definitive neural stem cell proliferation from the adult spinal cord. Stem Cells. 35 (2), 485-496 (2017).
- Sachewsky, N., et al. Primitive neural stem cells in the adult mammalian brain give rise to GFAP-expressing neural stem cells. Stem Cell Reports. 2 (6), 810-824 (2014).
- Mothe, A., Tator, C. H. Isolation of neural stem/progenitor cells from the periventricular region of the adult rat and human spinal cord. Journal of Visualized Experiments. (99), (2015).
- Absher, M. Hemocytometer counting. Tissue Culture. , 395-397 (1973).
- Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay. Journal of Visualized Experiments. (45), (2010).
- Xiong, L., et al. Preconditioning with isoflurane produces dose-dependent neuroprotection via activation of adenosine triphosphate-regulated potassium channels after focal cerebral ischemia in rats. Anesthesia and Analgesia. 96 (1), 233-237 (2003).
- Metz, G. A., Merkler, D., Dietz, V., Schwab, M. E., Fouad, K. Efficient testing of motor function in spinal cord injured rats. Brain Research. 883 (2), 165-177 (2000).
- Hamers, F. P., Koopmans, G. C., Joosten, E. A. CatWalk-assisted gait analysis in the assessment of spinal cord injury. Journal of Neurotrauma. 23 (3-4), 537-548 (2006).
