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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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=Reactive+astrocytes+protect+tissue+and+preserve+function+after+spinal+cord+injury.">Reactive astrocytes protect tissue and preserve function after spinal cord injury.</a> Journal of Neuroscience. 24 (9), 2143-2155 (2004).</li><li>Cheriyan, 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=Spinal+cord+injury+models:+a+review.">Spinal cord injury models: a review.</a> Spinal Cord. 52 (8), 588-595 (2014).</li><li>Deleyrolle, L. P., 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,+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?...

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 border="0" cellpadding="0" cellspacing="0" width="815">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Agricola Retractor</td>
			<td style="width:201px;">Fine Science Tools</td>
			<td style="width:197px;">17005-04</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Moria Vannas-Wolff Spring Scissors (Curved)</td>
			<td style="width:201px;">Fine Science Tools</td>
			<td style="width:197px;">15370-50</td>
			<td style="width:215px;">Customize when ordering to get blunted tips</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Graefe Forceps (Straight, 1x2 Teeth)</td>
			<td style="width:201px;">Fine Science Tools</td>
			<td style="width:197px;">11053-10</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Extra Fine Graefe Forceps (Curved, Serrated)</td>
			<td style="width:201px;">Fine Science Tools</td>
			<td style="width:197px;">11152-10</td>
			<td style="width:215px;">Or any other forceps for suturing</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Hartman Hemostats (Straight)</td>
			<td style="width:201px;">Fine Science Tools</td>
			<td style="width:197px;">13002-10</td>
			<td style="width:215px;">Or any other appropriate for suturing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Scalpel Handle #3</td>
			<td style="width:201px;">Fine Science Tools</td>
			<td style="width:197px;">10003-12</td>
			<td style="width:215px;">Or any other appropriate</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:203px;">Hair clippers</td>
			<td style="width:201px;">amazon.ca</td>
			<td style="width:197px;">https://www.amazon.ca/Wahl-Professional-8685-Classic-Clipper/dp/B00011K2BA</td>
			<td style="width:215px;">or any other appropriate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Stereotaxic instrument</td>
			<td style="width:201px;">Stoeling</td>
			<td align="right">51500</td>
			<td style="width:215px;">or any other appropriate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Buprenorphine</td>
			<td style="width:201px;"></td>
			<td style="width:197px;"></td>
			<td>or any appropirate sanctioned my animal care facility</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Meloxicam</td>
			<td style="width:201px;"></td>
			<td style="width:197px;"></td>
			<td style="width:215px;">or any appropriate sanctioned by animal care facility</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:203px;">Tears Naturale P.M.</td>
			<td style="width:201px;">Alcon</td>
			<td style="width:197px;">https://www.amazon.ca/Alcon-Tear-Gel-Liquid-Eye-Gel/dp/B00HHXGUXE</td>
			<td style="width:215px;">or any other appropriate</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Isoflurane</td>
			<td style="width:201px;">Baxter International Inc</td>
			<td style="width:197px;">DIN 02225875</td>
			<td style="width:215px;">or any other appropriate for anesthesia</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:203px;">Q-tips Cottom Swabs</td>
			<td style="width:201px;">amazon.ca</td>
			<td style="width:197px;">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&#38;psc=1&#38;refRID=JC16N542KVRF2N62N3DS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Cotton Gauze</td>
			<td style="width:201px;">Fisher Scientific</td>
			<td>13-761-52</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">30G Needles</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td align="right" style="width:197px;">305106</td>
			<td>For Injury</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">25G Needles</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td align="right" style="width:197px;">305122</td>
			<td>For Drug injections</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">1mL Syringes</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td align="right" style="width:197px;">3090659</td>
			<td>for drug injections</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">3mL Syringes</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td align="right" style="width:197px;">309657</td>
			<td>for fluid injections</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">4-0 Suture</td>
			<td style="width:201px;">uoftmedstore.com</td>
			<td>2297-VS881</td>
			<td>for skin suturing</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">6-0 Suture</td>
			<td style="width:201px;">uoftmedstore.com</td>
			<td>VS889</td>
			<td>for muscle suturing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Polysporin ointment</td>
			<td style="width:201px;">amazon.ca</td>
			<td align="right">102051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Isoflurane Vaporizer</td>
			<td style="width:201px;">VetEquip</td>
			<td align="right" style="width:197px;">901806</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">15mL conical tubes</td>
			<td style="width:201px;">ThermoFisher</td>
			<td></td>
			<td>Any appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Petri Dishes</td>
			<td style="width:201px;">ThermoFisher</td>
			<td style="width:197px;"></td>
			<td>any appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Trypan Blue</td>
			<td style="width:201px;">ThermoFisher</td>
			<td style="width:197px;"></td>
			<td>Any</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Hemocytometer</td>
			<td style="width:201px;">ThermoFisher</td>
			<td style="width:197px;"></td>
			<td>Any appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Centrifuge</td>
			<td style="width:201px;">ThermoFisher</td>
			<td style="width:197px;"></td>
			<td>Any appropriate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Standard Dissection Tools</td>
			<td style="width:201px;">Fine Science Tools</td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Dissection Microscope</td>
			<td style="width:201px;">Zeiss</td>
			<td style="width:197px;">Stemi 2000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Counting Microscope</td>
			<td style="width:201px;">Olympus</td>
			<td style="width:197px;">CKX41</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Neural Basal-A Medium</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:197px;">10888-022</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">B27</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:197px;">17404-044</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Penicillin- Streptomycin</td>
			<td style="width:201px;">Gibco</td>
			<td align="right" style="width:197px;">15070</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">L- Glutamine</td>
			<td style="width:201px;">Gibco</td>
			<td align="right" style="width:197px;">25030</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">DMEM</td>
			<td style="width:201px;">Invitrogen</td>
			<td align="right" style="width:197px;">12100046</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">F12</td>
			<td style="width:201px;">Invitrogen</td>
			<td align="right" style="width:197px;">12700075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">30% Glucose</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">G6152</td>
			<td>1M- 9.01g in 100mL dH2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">1M Glucose</td>
			<td style="width:201px;"></td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">7.5% NaHCO3</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">S5761</td>
			<td>155mM- 1.30g in 100mL dH2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">155mM NaHCO3</td>
			<td style="width:201px;"></td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">1M HEPES</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">H3375</td>
			<td>23.83 g in 100mL dH2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Apo-Transferrin</td>
			<td style="width:201px;">R&#38;D Systems</td>
			<td style="width:197px;">3188-AT</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Putrescine&#160;</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">P7505</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Insulin</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">I5500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Selenium</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">S9133</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Progesterone</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">P6149</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Papain Dissociation System&#160;</td>
			<td style="width:201px;">Worthington Biochemical Corporation</td>
			<td style="width:197px;">PDS</td>
			<td>1 vial of papain can be used for 2 samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Epidermal Growth Factor</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:197px;">PMG8041</td>
			<td>Powder reconstituted with 1mL Hormone Mix and aliquoted into 20uL vials to be stored in freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Fibroblast Growth Factor</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:197px;">PHG0226</td>
			<td>Powder reconstituted with 0.5mL Hormone Mix and aliquoted into 20uL vials to be stored in freezer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Heparin</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">H3149</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Leukemia Inhibitory Factor</td>
			<td style="width:201px;">In House</td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Trypan Blue</td>
			<td style="width:201px;"></td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Hemocytometer</td>
			<td style="width:201px;"></td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">24 well Plates</td>
			<td style="width:201px;">NUNC</td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">2M NaCl</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">S5886</td>
			<td>11.69g in 100mL dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">1M KCL</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">P5405</td>
			<td>7.46g in 100mL dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">1M MgCl2</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">M2393</td>
			<td>20.33g in 100mL dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">108mM CaCl2</td>
			<td style="width:201px;">Sigma&#160;</td>
			<td style="width:197px;">C7902</td>
			<td>1.59g in 100mL dH2O</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) ?...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.8K 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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

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 ...

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 ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

侧向液压冲击:创伤性脑损伤小鼠模型

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

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 ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

小鼠完全转型脊髓损伤的诱导

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

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

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此视频中的章节

从胚胎脊髓连合神经的解剖和文化

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

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

侧向液压冲击:创伤性脑损伤小鼠模型

一个

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

拉米内切手术和脊髓窗口植入小鼠

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

小鼠完全转型脊髓损伤的诱导

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

此视频中的章节

从胚胎脊髓​​连合神经的解剖和文化

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

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

侧向液压冲击:创伤性脑损伤小鼠模型

一个

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

拉米内切手术和脊髓窗口植入小鼠

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

小鼠完全转型脊髓损伤的诱导

从胚胎脊髓连合神经的解剖和文化