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。

该议定书已获多伦多大学动物保育委员会批准, 并符合 "实验动物护理及使用指南" (2 版, 加拿大动物保育委员会, 2017).
1. 最小脊髓损伤手术
注: 手术前确保所有手术器械和材料均采用适当的方法进行灭菌 (图 1A)。
<ol><li>建立一个胸支拱通过卷起第4-5 广场的纱布, 并把他们一起在中间, 以获得一卷固定的纱布。</li>
	<li>将鼠标放置在感应腔内, 用5% 异氟醚启动麻醉。确认没有脚趾捏反射, 然后继续, 以及整个过程。由于手术可以采取30分钟以上的完成, 优化最小的异氟醚暴露 (5 分钟5% 和剩余时间在 2–3%)。</li>
	<li>将鼠标转移到附着在2–3% 异氟醚维护剂量的麻醉机上的鼻锥上。使用剪发刀从背部的动物背部到颈部/耳朵上去除毛皮, 以暴露一个宽矩形区域的皮肤进行手术。</li>
	<li>把它的鼻子放在附着的鼻锥上, 用耳棒稳定头骨, 将鼠标转移到立体定向的仪器上。当鼠标在立体定向装置中固定后, 在放置耳棒和下背部至2–3% 时, 将异氟醚保持在5%。</li>
	<li>管理术前镇痛药物腹腔-Meloxicam (2.0 毫克/千克)。在麻醉时, 在睁开的老鼠眼上慷慨地涂抹眼部润滑, 防止角膜干燥。</li>
	<li>将胸支弓放在小鼠腹部下面, 同时在尾部的底部轻轻拉直鼠标的身体和脊柱。使用实验室标签胶带, 以确保在星状位置的尾巴和所有伸展四肢。一旦安全, 推胸支持弓 rostrally, 从鼠标腹部向上胸部-以支撑胸椎 (图 1B)。</li>
	<li>通过消毒步骤1.3 中用70% 乙醇制备的毛皮修剪皮肤区域, 然后用聚维酮碘, 制备无菌手术场。重复两次。使用无菌的手术悬垂, 以保持广泛的无菌领域。</li>
	<li>使用 #10 手术刀刀片, 使垂直切口平行于动物的纵轴从两个肩胛骨的中点到胸椎的曲率。收回皮肤暴露软组织和脊柱轮廓 (图 1C)。</li>
	<li>识别肩胛脂肪垫的下边界 (这界定 T4/5 椎体)。用同样的 #10 刀片, 小心, 但与力量, 切断沿两侧的椎骨 T5–T8/9 分离背部肌肉肌腱从专栏。</li>
	<li>将拉钩的牙齿插入脊柱两侧的切口部位。通过扩大拉钩来调整曝光量, 以充分提升脊柱, 而不会对缩回的肌肉层施加太多的压力 (图 1D)。</li>
	<li>在手术显微镜下, 仔细清理脊柱上的残余肌肉和其他软组织, 以暴露椎体骨 (图 1E)。通过用齿形钳紧握椎骨的棘突, 并稍微向上和向下移动, 以确定将被切除的椎骨。 注意: 这应允许识别椎间关节, 并暴露在所关心的椎骨下面的小开口 (椎间孔)。</li>
	<li>将弯曲钝剪刀的一头插入到暴露的椎间孔的两侧, 将椎板尾部切除, 并在双边上切开连接的椎体关节。</li>
	<li>抬起叶片向上和切断的上部附着的叶片, 以隔离和删除骨。 注意: 这将暴露完整的硬脑膜囊包含脊髓 (图 1F)。可能有过多的出血, 可以控制通过放置预制1厘米 x 1 cm 纱布到受影响的地区和/或洗涤与无菌 PBS。</li>
	<li>以背中线静脉作为标志性, 插入一个45°弯曲轴 30 G 针尖 (与针斜角向上) 进入侧表面的脊髓 (大约1毫米深) 在大约0.5 毫米侧向两侧的中线。</li>
	<li>将针从尾侧移动到延髓 (平行于中线), 以便将针的整个长度插入线中。通过输入路径 (图 1G) 来删除针。 注意: 不应该有出血, 但在这一步可以看到脊椎组织肿胀。</li>
	<li>要关闭伤口, 取出牵引器, 并缝合两侧的背部肌肉的伤害在中线使用6-0 可吸收缝合。使用4-0 无菌丝缝合, 随后关闭上覆皮肤。</li>
	<li>将抗生素软膏涂抹在表面缝合皮肤的顶端, 以防止使用棉签尖端的术后感染。术后镇痛0.1 毫克/千克的丁丙诺啡皮下连同液体 (1 毫升乳酸的解决方案)。</li>
	<li>关闭异氟醚的蒸发器和氧气。将鼠标从立体定向装置中取出, 并放置在没有寝具的干净笼子里。</li>
	<li>将鼠标放在笼子里大约30厘米远的地方, 从热灯中恢复麻醉, 并在醒来后监测行为。老鼠应该在5–10分钟内醒来, 在醒来后有可能麻痹和/或尾巴虚弱的后肢功能。</li>
	<li>监测鼠标的行为在未来几天, 并提供适当的术后护理和镇痛药 (即, 饲料, 乳酸的解决方案液体皮下, 0.1 毫克/千克丁丙诺啡2x 每天2–3天, 并适当的体重监测) 根据当地动物设施条例和个人情况, 恢复可能有所不同。</li>
</ol>2. 干细胞化验解剖
注意: 在整个过程中遵循适当的无菌组织培养程序。
<ol><li>酶解制剂
	<ol>
<li>将32毫升的厄尔平衡盐溶液 (EBSS) 加入到含有玻璃瓶的白蛋白 ovomucoid 抑制剂中, 然后轻轻地涡旋直至溶解。</li>
		<li>加入5毫升的 EBSS 到前便餐木瓜蛋白酶玻璃瓶和地方在37°c 水浴溶化。解决方案将变得清晰 (解决方案 1)。</li>
		<li>采取500µL 的 EBSS 和添加到前便餐 DNase I 玻璃瓶获得1毫克/毫升浓度 (溶液 2)。将瓶子来回倾斜, 然后将250µL 的混合物添加到溶液1中, 轻轻地搅拌。 注意: 所有的解决方案 (解决方案1和 2) 足够处理两个组织。如果处理更多的组织, 适应更多的解决方案准备。</li>
	</ol></li>
	<li>组织准备和解剖
	<ol>
<li>将实验室擦拭用异氟醚浸泡在老鼠笼中, 并允许 2-3 分钟的蒸气完全麻醉鼠标。击倒后, 从笼子中取出鼠标, 并确认没有脚趾捏反射。</li>
		<li>用钝金属物体 (即金属笼卡) 进行颈椎脱位弄死动物。用70% 乙醇将安乐死的动物从颈部向中背部喷洒。</li>
		<li>在头骨的底部, 使用剪刀切断头部, 并丢弃到一个生物袋。在皮肤沿背部的整个长度进行中线切口, 以暴露肌肉和椎骨轮廓 (图 2A)。</li>
		<li>抬起每个肩胛骨的内侧部分 (i., 肩胛骨-通过上覆脂肪垫识别), 并用剪刀剪掉软组织 (肩胛和椎骨之间)。完全分离, 以暴露基础脊椎柱。</li>
		<li>根据脊柱的曲率, 将同一把剪刀插入上胸椎孔, 通过切断附着的肋骨、肌肉和内脏来隔离椎柱 (图 2B)。</li>
		<li>将剪刀插入脊椎椎孔/开口处, 并在板的两侧 (背表面) 切开椎间关节。特别小心, 不要损坏完整的绳子。继续此过程, 直到所需的线的水平和/或长度被暴露。</li>
		<li>在将脊髓组织转移到有规律的人工脑脊液的培养皿之前, 小心地切断从脐带侧面延伸的脊髓神经。把样品放在冰上 (图 2C)。</li>
		<li>在解剖显微镜下, 切断脊髓组织, 包括2毫米延髓和尾鳍到受伤部位。使用两对钳, 轻轻地将脐带沿背侧和腹侧裂隙纵向分开2半 (图 2C)。</li>
		<li>与 microscissors, 切出受伤的侧白色物质。将该片放入15毫升锥形管中。</li>
		<li>用镊子将脊髓的一半用钳夹住, 用另一对细钳沿着脊髓的 rostrocaudal 范围来挑逗和去除白质, 以隔离所需的脑室区域。重复另一半脊髓。</li>
		<li>将脑室组织块放入一个单独的15毫升锥形管中。使用 microscissors 和轻轻地切碎的组织对壁的锥形管。 注: 组织离解修改是从木瓜蛋白酶离解系统协议23和以前描述的组织准备程序24。</li>
		<li>添加2.5 毫升新的解决方案 1 (步骤 2.1.3) 到组织样本和放置在一个摇杆37°c 30 分钟。</li>
		<li>用1000µL 吸管尖轻轻 triturate 溶液中的组织。离心机的多云细胞悬浮在 300 x g 5 分钟的 RT。</li>
		<li>准备解决方案3与2.7 毫升的厄尔的平衡盐溶液, 300 µL 的 ovomucoid 抑制剂溶液 (步骤 2.1.1) 和150µL DNase I 溶液 (步骤 2.1.3)。</li>
		<li>从上一步 (步骤 2.2.14) 中, 丢弃从步骤 (步骤 2.2.13) 和并用重悬1.5 毫升溶液3中的上清。</li>
		<li>将这个新的细胞悬浮层 (步骤 2.2.15) 放在5毫升的 ovoimucoid 抑制剂溶液 (步骤 2.1.1) 上, 在一个新的15毫升圆锥管中创建一个不间断的密度梯度。离心机在 300 x g 5 分钟的 RT。</li>
		<li>在2毫升的 neurobasal 介质中丢弃上清和并用重悬。离心机在 300 x g 3 分钟的 RT。</li>
		<li>在1毫升的 neurobasal 介质中丢弃上清和并用重悬。过滤悬浮使用20µm 细胞过滤器跟随4毫升媒介为总容量5毫升。</li>
	</ol></li>
	<li>电镀
	<ol>
<li>用表皮生长因子 (20 干细胞/毫升)/成纤维细胞生长因子 (20 neurobasal)/肝素 (2 µg/毫升) [用于确定神经干细胞] 或 leukemina 抑制因子 (10 ng/毫升), 对原始神经干细胞进行补充, 制备培养培养基以促进生长。在25毫升的组织培养瓶中加入10毫升的培养基。</li>
		<li>使用 hemocytometer 和台盼蓝染料计算悬浮液中的细胞数 (步骤 2.2.18)25。一旦计算, 将细胞添加到组织培养烧瓶中 (步骤 2.3.1) 到10细胞/µL 的克隆密度, 并将组织培养瓶与添加的细胞放入孵化器中24小时 (37 °c, 95% 湿度, 5% CO2)。 注: 计数技术的一个例子是: 采取10µL 的细胞悬浮和混合10µL 的台盼蓝染料。将此混合物的10µL 加入 hemocytometer。在10X 光显微镜下, 计算活细胞数, 并对 4 x 4 象限内4以内的所有细胞求和。使用公式: 10万/[(x cells/4) x 2 x 10 x 1000] 使细胞悬浮量在每25毫升组织培养瓶中被镀, 以确保克隆密度 (10 细胞/µL)。</li>
		<li>24小时后, 轻轻地旋转烧瓶, 倒入15毫升圆锥管中的内容。离心机在 300 x g 5 分钟的 RT。</li>
		<li>放弃上清, 并在新鲜的 neurobasal 培养基中重新悬浮细胞。</li>
		<li>重复步骤2.3.2 计算24井组织培养板中的悬浮细胞和板细胞的数量, 其中500µL 各自的丝裂原补充培养基 (经济适用房为确定的国安生长和原始的神经干细胞生长的 LIF)。 注: 使用所适应的公式 (5000/[X cells/4) x 2 x 10 x 1000) 计算在每一个含有500µL 介质的井中所镀的细胞悬浮体的体积。</li>
		<li>在孵化器 (37 °c, 95% 湿度, 5% CO2) 中放置含有细胞的板块, 生长7天。</li>
	</ol></li>
	<li>计数
	<ol>
<li>经过7天的文化, 删除板和视野下的光显微镜。通过计数 > 80 µm 直径的球形菌落来量化 neurospheres pNSCs 文化的 dNSCs 和 > 50 µm 直径。 注: 如果需要, neurospheres 可以进一步传代26或区分。如果不再需要细胞, 将加速过氧化氢添加到油井 (约1毫升), 以20分钟, 使培养基的颜色变黄。然后丢弃。</li>
	</ol></li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+expansion,+and+differentiation+of+adult+Mammalian+neural+stem+and+progenitor+cells+using+the+neurosphere+assay.">Isolation, expansion, and differentiation of adult Mammalian neural stem and progenitor cells using the neurosphere assay.</a> Neural Cell Transplantation: Methods and Protocols. , 91-101 (2009).</li><li>Singec, I., 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=Defining+the+actual+sensitivity+and+specificity+of+the+neurosphere+assay+in+stem+cell+biology.">Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.</a> Nature Methods. 3 (10), (2006).</li><li>Mignone, J. L., Kukekov, V., Chiang, A. S., Steindler, D., Enikolopov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+and+progenitor+cells+in+nestin-GFP+transgenic+mice.">Neural stem and progenitor cells in nestin-GFP transgenic mice.</a> The Journal of Comparative Neurology. 469 (3), 311-324 (2004).</li><li>Xu, W., 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=Myelin+basic+protein+regulates+primitive+and+definitive+neural+stem+cell+proliferation+from+the+adult+spinal+cord.">Myelin basic protein regulates primitive and definitive neural stem cell proliferation from the adult spinal cord.</a> Stem Cells. 35 (2), 485-496 (2017).</li><li>Sachewsky, N., 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=Primitive+neural+stem+cells+in+the+adult+mammalian+brain+give+rise+to+GFAP-expressing+neural+stem+cells.">Primitive neural stem cells in the adult mammalian brain give rise to GFAP-expressing neural stem cells.</a> Stem Cell Reports. 2 (6), 810-824 (2014).</li><li>Mothe, A., Tator, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neural+stem/progenitor+cells+from+the+periventricular+region+of+the+adult+rat+and+human+spinal+cord.">Isolation of neural stem/progenitor cells from the periventricular region of the adult rat and human spinal cord.</a> Journal of Visualized Experiments. (99), (2015).</li><li>Absher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemocytometer+counting.">Hemocytometer counting.</a> Tissue Culture. , 395-397 (1973).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay.">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> Journal of Visualized Experiments. (45), (2010).</li><li>Xiong, 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=Preconditioning+with+isoflurane+produces+dose-dependent+neuroprotection+via+activation+of+adenosine+triphosphate-regulated+potassium+channels+after+focal+cerebral+ischemia+in+rats.">Preconditioning with isoflurane produces dose-dependent neuroprotection via activation of adenosine triphosphate-regulated potassium channels after focal cerebral ischemia in rats.</a> Anesthesia and Analgesia. 96 (1), 233-237 (2003).</li><li>Metz, G. A., Merkler, D., Dietz, V., Schwab, M. E., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+testing+of+motor+function+in+spinal+cord+injured+rats.">Efficient testing of motor function in spinal cord injured rats.</a> Brain Research. 883 (2), 165-177 (2000).</li><li>Hamers, F. P., Koopmans, G. C., Joosten, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CatWalk-assisted+gait+analysis+in+the+assessment+of+spinal+cord+injury.">CatWalk-assisted gait analysis in the assessment of spinal cord injury.</a> Journal of Neurotrauma. 23 (3-4), 537-548 (2006).</li></ol>

作者没有什么可透露的。

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, 并且不应该太慢 (每2–3的呼吸少于1次) 或大量吃力 (即喘气)。麻醉的维持剂量可以从2–3% 降低, 以防止因长时间麻醉而导致的死亡, 并注意到接受了较低剂量的小鼠。
在手术期间, 在脊柱两侧进行肌肉切口时要格外小心。确保切口是深的, 刀片是角度内侧, 使刀片的边缘在肌肉切开时在骨椎骨上休息。如果刀片向外倾斜, 则有可能因血管切开而流血过多。研究人员在执行椎板切除术时也应格外注意, 以避免将损伤脊髓的剪刀深钓, 造成不必要的组织损伤和功能缺陷。对这些实验的适当控制是一个 "只椎板" 组 (无损伤), 这将使比较的激活神经干细胞归因于针轨伤害, 而不是只能由椎板切除的结果。我们已经表明, 单板切除可以导致一个小的, 虽然微不足道的增加的神经干细胞激活, 显示了干细胞数字从脑室地区在病变级别21。仅椎板切除不会导致从脑室地区延髓或尾鳍到椎板切除术的干细胞数增加, 在椎板切除的白色物质的培养中没有发现 neurospheres。另外, 在执行椎板切除术时, 要注意将背板移除一片, 以允许广泛暴露脐带。这将 (1) 允许足够的访问, 以执行最小的针轨伤害的侧脊髓, (2) 防止骨段留在后面, 并造成继发损伤后, 关闭和恢复运动的鼠标。如果整个叶片不作为一个片断, 或在椎板的两侧突出的尖锐骨段被观察, 使用仪器 (如齿钳和弯曲剪刀), 以消除这些碎片之前缝合。
在手术结束时, 研究人员在肌肉层应用缝线时应格外小心。一条缝合 (双打结) 应放置尾部到椎板切除/损伤的水平, 使缝合位于完整的椎骨的顶端。这是为了防止任何次要损害 , 可能造成的肌合接触暴露脐带时 , 鼠标移动后 , 恢复。此外 , 椎板切除术 / 损伤后的肌合可以作为在隔离脊髓进行分析时进行 SCI 的里程碑。在解剖损伤的脊髓区域时应注意避免损害受损区域的结构, 以便在解剖显微镜下进行精细解剖时保持可辨认性。对于干细胞化验, 重要的是要进行细胞微丸的机械研磨, 以避免产生气泡, 可以增加细胞死亡。pNSC 衍生的 neurospheres 更敏感的碎片重, 生长条件-过度研磨和长期暴露在酶解-相对于 dNSC 的文化。pNSC 衍生球比 dNSCs 更紧凑、更小。鉴于 pNSCs 的稀有性, 我们建议每样样品至少隔离24万个细胞。
最小 SCI 模型是研究损伤后细胞事件 (如内源性神经干细胞活化) 的理想方法, 但不允许对功能性损伤的研究。如前所述, 从针道损伤中恢复的小鼠没有明显的行为缺陷持续存在, 因此, 不能对小鼠进行评估以提高功能性结局的治疗干预效果。再生医学的一个重要方面是, 治疗应该不仅促进组织修复 (可以使用这个模型评估, 结合干细胞化验, 血统追踪和免疫组化/免疫荧光), 但还应在适用的情况下, 使用行为范式证明相关的功能改进。许多行为任务用于测试胸部损伤模型中的功能结果, 如足部断层试验和低音、贝蒂、Breshnan (BBB) 开放场运动规模28, 都不够灵敏, 无法检测到我们最小的可测量的赤字。SCI 模型。为了克服这一缺点, 人们可以利用更灵敏的数字计分系统 (例如, 台步) 来测量包括步态分析在内的粗、细后肢运动参数的多个参数, 这可能会检测到最小最低伤害模型29造成的赤字。
该方法还可用于研究内源性神经干细胞活化作为颈髓损伤模型的潜在治疗。颈椎 SCI 是临床上最相关的模型, 我们建议将这种损伤模型适应更高的椎段, 可以洞察神经干细胞及其子代的反应是否有区域差异 (动力学、迁移、分化) 和病变是否会导致更深刻和可测量的功能性缺陷。目前使用的小鼠颈椎损伤模型 (如横断、夹压和/或挫伤) 需要大量的术后护理, 包括需要手动表达膀胱, 并不断监测鼠标的呼吸。将最小损伤模型适应于颈椎脊髓可降低与其他颈椎 SCI 模型相关的死亡率、发病率和术后护理, 并允许检查药物/小分子和/或康复的有效性。神经恢复的结果。我们的伤害模型也可以被改编成在脐带的不同部位 (即背侧或侧面柱) 和/或更大的伤害更深的穿透和/或使用较大的针 (较小的量规) 造成最小的伤害。这使得研究者能够控制/操纵病变的类型和大小, 从而对细胞和功能/行为恢复进行相应的评估。
小鼠最小损伤模型允许使用转基因动物模型。老鼠模型能在损伤之前标记内源性干细胞和/或祖细胞。这可以让研究者跟踪损伤后的这些预先标记细胞的命运, 评估神经前体细胞的增殖、迁移和损伤后的分化--可能有助于神经修复。

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。

<table><tbody><tr><td>Agricola Retractor</td><td>Fine Science Tools</td><td>17005-04</td><td></td></tr><tr><td>Moria Vannas-Wolff Spring Scissors (Curved)</td><td>Fine Science Tools</td><td>15370-50</td><td>Customize when ordering to get blunted tips</td></tr><tr><td>Graefe Forceps (Straight, 1 x 2 Teeth)</td><td>Fine Science Tools</td><td>11053-10</td><td></td></tr><tr><td>Extra Fine Graefe Forceps (Curved, Serrated)</td><td>Fine Science Tools</td><td>11152-10</td><td>Or any other forceps for suturing</td></tr><tr><td>Hartman Hemostats (Straight)</td><td>Fine Science Tools</td><td>13002-10</td><td>Or any other appropriate for suturing</td></tr><tr><td>Scalpel Handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td>Or any other appropriate</td></tr><tr><td>Hair clippers</td><td>amazon.ca</td><td>https://www.amazon.ca/Wahl-Professional-8685-Classic-Clipper/dp/B00011K2BA</td><td>or any other appropriate</td></tr><tr><td>Stereotaxic instrument</td><td>Stoeling</td><td>51500</td><td>or any other appropriate</td></tr><tr><td>Buprenorphine</td><td></td><td></td><td>or any appropirate sanctioned my animal care facility</td></tr><tr><td>Meloxicam</td><td></td><td></td><td>or any appropriate sanctioned by animal care facility</td></tr><tr><td>Tears Naturale P.M.</td><td>Alcon</td><td>https://www.amazon.ca/Alcon-Tear-Gel-Liquid-Eye-Gel/dp/B00HHXGUXE</td><td>or any other appropriate</td></tr><tr><td>Isoflurane</td><td>Baxter International Inc</td><td>DIN 02225875</td><td>or any other appropriate for anesthesia</td></tr><tr><td>Q-tips Cottom Swabs</td><td>amazon.ca</td><td>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&amp;amp;psc=1&amp;amp;refRID=JC16N542KVRF2N62N3DS</td><td></td></tr><tr><td>Cotton Gauze</td><td>Fisher Scientific</td><td>13-761-52</td><td></td></tr><tr><td>30 G Needles</td><td>Becton Dickinson</td><td>305106</td><td>For Injury</td></tr><tr><td>25 G Needles</td><td>Becton Dickinson</td><td>305122</td><td>For Drug injections</td></tr><tr><td>1 mL Syringes</td><td>Becton Dickinson</td><td>3090659</td><td>for drug injections</td></tr><tr><td>3 mL Syringes</td><td>Becton Dickinson</td><td>309657</td><td>for fluid injections</td></tr><tr><td>4-0 Suture</td><td>uoftmedstore.com</td><td>2297-VS881</td><td>for skin suturing</td></tr><tr><td>6-0 Suture</td><td>uoftmedstore.com</td><td>VS889</td><td>for muscle suturing</td></tr><tr><td>Polysporin ointment</td><td>amazon.ca</td><td>102051</td><td></td></tr><tr><td>Isoflurane Vaporizer</td><td>VetEquip</td><td>901806</td><td></td></tr><tr><td>15 mL conical tubes</td><td>ThermoFisher</td><td></td><td>Any appropriate</td></tr><tr><td>Petri Dishes</td><td>ThermoFisher</td><td></td><td>any appropriate</td></tr><tr><td>Trypan Blue</td><td>ThermoFisher</td><td></td><td>Any</td></tr><tr><td>Hemocytometer</td><td>ThermoFisher</td><td></td><td>Any appropriate</td></tr><tr><td>Centrifuge</td><td>ThermoFisher</td><td></td><td>Any appropriate</td></tr><tr><td>Standard Dissection Tools</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Dissection Microscope</td><td>Zeiss</td><td>Stemi 2000</td><td></td></tr><tr><td>Counting Microscope</td><td>Olympus</td><td>CKX41</td><td></td></tr><tr><td>Neural Basal-A Medium</td><td>Invitrogen</td><td>10888-022</td><td></td></tr><tr><td>B27</td><td>Invitrogen</td><td>17404-044</td><td></td></tr><tr><td>Penicillin- Streptomycin</td><td>Gibco</td><td>15070</td><td></td></tr><tr><td>L- Glutamine</td><td>Gibco</td><td>25030</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>12100046</td><td></td></tr><tr><td>F12</td><td>Invitrogen</td><td>12700075</td><td></td></tr><tr><td>30% Glucose</td><td>Sigma</td><td>G6152</td><td>1 M, 9.01 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>1 M Glucose</td><td></td><td></td><td></td></tr><tr><td>7.5% NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td>155 mM, 1.30 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>155 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>1 M HEPES</td><td>Sigma</td><td>H3375</td><td>23.83 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Apo-Transferrin</td><td>R&amp;amp;D Systems</td><td>3188-AT</td><td></td></tr><tr><td>Putrescine&amp;nbsp;</td><td>Sigma</td><td>P7505</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I5500</td><td></td></tr><tr><td>Selenium</td><td>Sigma</td><td>S9133</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P6149</td><td></td></tr><tr><td>Papain Dissociation System&amp;nbsp;</td><td>Worthington Biochemical Corporation</td><td>PDS</td><td>1 vial of papain can be used for 2 samples</td></tr><tr><td>Epidermal Growth Factor</td><td>Invitrogen</td><td>PMG8041</td><td>Powder reconstituted with 1mL Hormone Mix and aliquoted into 20uL vials to be stored in freezer</td></tr><tr><td>Fibroblast Growth Factor</td><td>Invitrogen</td><td>PHG0226</td><td>Powder reconstituted with 0.5 mL Hormone Mix and aliquoted into 20 &amp;mu;L vials to be stored in freezer</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>Leukemia Inhibitory Factor</td><td>In House</td><td></td><td></td></tr><tr><td>Trypan Blue</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>24 well Plates</td><td>NUNC</td><td></td><td></td></tr><tr><td>2 M NaCl</td><td>Sigma</td><td>S5886</td><td>11.69 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>1 M KCL</td><td>Sigma</td><td>P5405</td><td>7.46 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>1 M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M2393</td><td>20.33 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>108 mM CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma&amp;nbsp;</td><td>C7902</td><td>1.59 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr></tbody></table>

a neurosphere assay to evaluate endogenous neural stem cell activation in a mouse model of minimal spinal cord injury

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。

手术后, 小鼠应经历最小的运动缺陷, 其中可能包括尾部和可能的后肢麻痹24小时。在这段时间后, 小鼠不应该经历后肢麻痹和/或麻痹和最小的步态变化。
图 3显示了最小脊髓损伤后5天干细胞试验的代表性结果。dNSC 衍生 neurospheres (生长于经济适用房) 的绝对数量大于损伤后 pNSC 衍生 neurospheres (生长于 LIF) 的数量 (分别为图 3A和3B)。更高功率图像的确定性干细胞 (图 3C) 和原始干细胞 (图 3D) 显示不同的外观, 这些不同的殖民地与明确的 neurospheres 是较大的直径 (≥80µm), 通常有一个很大的黑暗中心。原始 neurospheres 体积较小 (≥50µm), 细胞更紧密地包装。在图 3E (definitives) 和3F (原语) 中, 出现来自受损脐带不同部位的 neurospheres 的代表性数字。图 3E显示, 最小的 SCI 导致了 neurospheres 从中央运河在受伤水平上的显著增加, 而延髓无损伤脐带的相对适度增加。
有趣的是, 明确的 neurospheres 可以从病变部位产生, 这个区域不包含干细胞形成细胞在椎板瘤单独的小鼠。图 3F显示了类似的增加 pNSCs 后损伤, 除了 pNSC neurospheres 在病灶部位。因此, 只有 dNSCs 能够迁移到受伤地点的这段时间课程后受伤。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57727/57727fig1v2.jpg"> 图 1: 外科手术.(A) 所需的一切布局, 编号供参考。(B) 一张在立体定向装置上的鼠标的图片, 其伸直的身体、脊柱和四肢摊开并贴在下面的胸腔支撑拱上。(C) 一张有垂直皮肤切口的老鼠图片, 露出肌肉层和脊柱轮廓。(D) 一张老鼠身体的图片, 经过肌肉的切口和接触和隔离脊柱与拉钩。注意绷紧的肌肉层, 缺乏撕裂。(E) 移除肌肉层的孤立小鼠脊柱的图片, 显示椎体的骨表面 (3 段)。这也显示了棘突的过程和可能的椎间关节和椎板的中间椎骨被删除。(F) 椎板切除术后可见脊髓的图片。标记为背中线静脉。(G) 在背中线静脉两侧有双侧针道损伤的小鼠图片。有一个特写插入的45°弯曲轴30克针。<a href="https://www.jove.com/files/ftp_upload/57727/57727fig1v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57727/57727fig2v2.jpg"> 图 2: 脊髓解剖.(a) 一张被斩首的老鼠与中线皮肤切口的图片, 揭露背部肌肉和椎骨轮廓 (B) 一张孤立的椎柱的图片, 显示延髓和尾鳍方向。第二张图片显示脐带隔离, 第三个是在培养皿中显示隔离脊髓。(C) 对脊髓进行精细解剖的图形表示, 将不同的段 (脑室、损伤区) 移除并分离培养。<a href="https://www.jove.com/files/ftp_upload/57727/57727fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57727/57727fig3v3.jpg"> 图 3: 在最小脊髓损伤后干细胞检测的代表性结果.(A) 受损伤脊髓培养的 dNSC neurospheres (经济适用房) 的数量大于 (B) 在同等密度下电镀时 pNSC 衍生 neurospheres (生长在 LIF) 的数量。(C、 D)高功率图像的 "典型" neurospheres 在 (C) dNSC 文化和 (D) pNSC 文化。(E) 针道损伤极小, 导致中央运河 dNSC neurospheres 的数量显著增加, 受伤程度以及中央运河延髓受伤, 以及受伤部位。(F) pNSC 衍生的 neurospheres 不能与未受伤的脐带隔绝, 但在中央运河 (受伤和延髓中央运河) 受伤后可以隔离。误差线表示平均值的标准误差。<a href="https://www.jove.com/files/ftp_upload/57727/57727fig3v3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury at JoVE.com

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

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

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。

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

a-neurosphere-assay-to-evaluate-endogenous-neural-stem-cell

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.9K 次观看.  University of Toronto. 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。

视频: A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

科研

发育生物学

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

神经科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

医学

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

产后神经球的分离和扩张 （P1+3） 小鼠神经原性球

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递:对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

NPC Neurosphere Assay: An In Vitro Assay to Assess the Migratory Response of Neural Precursor Cells from the Neurosphere

Source: Williams, M. et al. <a href="https://www.jove.com/v/56628/rapid-detection-neurodevelopmental-phenotypes-human-neural-precursor">Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs).</a>&#160;J. Vis. Exp. (2018)
This video demonstrates the neural precursor cell neurosphere migration assay&#8212;an in vitro assay to study the migratory response of neural precursor cells from the neurosphere&#8212;via microscopic analysis.

NPC 神经球检测:一种评估神经前体细胞来自神经球的迁移反应的体外检测

Source: Williams, M. et al. Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs).&#160;J. Vis. Exp. (2018)
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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

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从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

成年鼠神经干细胞的分离和扩大使用神经干含量

建立小鼠胚胎神经干细胞培养神经球含量

促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后

在脊髓损伤的实验挫伤型号神经干细胞移植

新生儿小鼠脊髓压迫损伤模型

胚胎小鼠神经干细胞的分离与培养

产后神经球的分离和扩张（P1+3）小鼠神经原性球

神经干细胞向大鼠和小鼠大脑的动脉内传递:对脑缺血的应用

NPC 神经球检测:一种评估神经前体细胞来自神经球的迁移反应的体外检测

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

摘要

探索更多视频

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

成年鼠神经干细胞的分离和扩大使用神经干含量

建立小鼠胚胎神经干细胞培养神经球含量

促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后

在脊髓损伤的实验挫伤型号神经干细胞移植

新生儿小鼠脊髓压迫损伤模型

胚胎小鼠神经干细胞的分离与培养

产后神经球的分离和扩张 （P1+3） 小鼠神经原性球

神经干细胞向大鼠和小鼠大脑的动脉内传递:对脑缺血的应用

NPC 神经球检测:一种评估神经前体细胞来自神经球的迁移反应的体外检测

产后神经球的分离和扩张（P1+3）小鼠神经原性球