该研究得到了生命之翼基金会 （WFL-IT-20/21）、欧盟下一代欧盟-国家恢复和复原力计划 （NRRP）-任务 4 第 2 部分、投资 n.1.4-CUP N.B83C22003930001（托斯卡纳健康生态系统-THE，辐条 8）和 Marina Romoli Onlus 的支持。本手稿仅反映作者的观点和意见，欧盟和欧盟委员会均不能被视为对此负责。数据和元数据可在 Zenodo 10.5281/zenodo 上找到。10433147.图像是使用 Biorender https://www.biorender.com/ 生成的。

脊髓器官型培养物中 h-SC-NES 细胞的长期植入

提交人无需声明任何利益冲突。

对于SCI患者，目前还没有有效的治疗方法。 已经测试了不同的方法，其中最有希望的方法之一是基于再生策略 - 细胞替换。目前，再生医学领域的进步要求人们提供新的平台来测试细胞移植的疗效和安全性，无论是单独使用还是与其他方法结合使用。他们的临床前验证对于进行进一步的临床研究至关重要。SC 器官型培养是研究神经退行性疾病、神经再生和神经发育不同方面以及研究新型治疗方法有效性的有用平台23。特别是，器官型培养物的特定特征（例如维持原始组织结构以及细胞和微环境组成）有利于揭示移植动力学（例如细胞植入、整合、分化和成熟）。
与已发表的方案一致，SC器官型切片可以在健康条件下在培养物中维持约2-3周，这限制了它们用于测试细胞治疗方案所需的长期研究和功能筛选。探索分化和成熟等重要过程，以确定 SC 组织内移植细胞的正确命运，需要长期监测。在动物模型的常见移植过程中，这些细胞过程至关重要。在临床前筛选阶段，模拟体内存在的许多特征的离体系统的可用性将有所帮助。
出于这个原因，在这项工作中，我们提出了一种最佳的长期（≥30 天）SC 器官型培养方法，该方法允许将活的 SC 切片维持长达 90 天，是其通常培养时间框架的三倍。此外，我们展示了稳定的 h-SC-NES 细胞在 SC 切片内植入，并且移植培养物可维持长达 30 天。我们通过观察 GFP 表达来监测细胞植入随时间的变化，以验证细胞存活率高达 DPT 30。30 DPT 后，我们评估细胞凋亡率。在文献中，已报道在 7 DPT 下 SC 切片中移植的 h-SC-NES 细胞凋亡的评估40。在这里，我们扩展了 DPT 30 的细胞凋亡分析，以比较相对于早期时间点 （DPT 7） 的凋亡率。我们发现我们的数据与文献一致，表明移植的 h-SC-NES 细胞如果保持在我们工作中优化的培养条件下，它们也会在以后的时间点存活。这种改进的长期 离体 平台单独使用和移植配置将帮助研究人员进行基于干细胞的SCI移植的临床前筛选。这将使他们能够确定最佳的细胞候选者，用于进一步的 体内 研究，促进移植的成功。此外，在初步筛选后，SC器官型切片也可以与 体内 研究并行使用，以确认和证实在动物模型中观察到的长期细胞动力学和行为，或支持机制研究。
我们的协议详细描述了如何生成这种长期器官型模型，但也应讨论一些关键步骤。关于SC器官型培养物的生成，在手术和培养的第一阶段存在一些挑战。执行良好的手术程序对于生成保持原始组织结构的切片至关重要。如果 SC 在分离过程中被破坏，切片可能会失去其典型的解剖结构，组织损伤会诱发过度的促炎损伤，从而导致不健康的状况和细胞死亡。手术中最具挑战性的阶段是从脊柱中取出皮下细胞瘤，并从孤立的皮下皮带中切除脑膜。这些步骤的成功取决于操作者的经验;因此，建议在开始实验之前进行一段时间的训练。
通过斩波器对 SC 进行冠状切片也是一个具有挑战性的阶段。隔离的 SC 应放置在完全垂直于刀片的切割台上。操作员还应将刀片垂直于切割台放置。这些预防措施对于确保在相同和不同实验之间产生可重复的切片是必要的。另一个重要问题是手术时间有限:整个切片生成过程必须需要 ~30 分钟。如果操作者在手术和切割上花费更多时间，SC组织将受到影响，这可能会影响培养的成功和实验的后续步骤。
一旦将切片放在培养膜上，正确喂养它们就很重要。GDNF 是维持组织恢复和存活所必需的。用切碎器切割对组织是创伤性的，因此，在切割后不久将切片放入冰冷的解剖培养基中，以清除多余的促炎和促死亡分子。然后，将切片用用 GDNF 改性的新鲜培养基放置在培养膜（细胞培养插入物）上，以促进更快的恢复和切片与膜的粘附。由于GDNF的半衰期短，因此在培养的第一周内应每天将GDNF添加到培养基中50,51。我们观察到，切片在培养的第一天需要持续存在 GDNF，以促进组织恢复和活力。无论如何，由于GDNF的存在对整个培养期都很重要，因此强烈建议不要在以后的时间点中断GDNF给药。
在培养的第一周，通过肉眼和显微镜观察切片也很重要。半透明的组织和边界的透明度是切片与膜和活组织正确粘附的标志。乍一看，坏死组织会呈现出极白的白色，而坏死区域在显微镜下会呈现深灰色。培养几周后，组织的形态可能会发生变化:细胞运动和组织与膜的粘附会影响这一过程。例如，我们观察到一些充满细胞的切片中中央管腔的丧失以及背角和腹角形态的丧失。这主要发生在较小的切片上，而其中大多数切片将保持接近原始切片的解剖结构。切片通常是从腰椎或胸部区域产生的，因为通过这种方式，它们可以具有适当的大小以随着时间的推移保持其原始的组织结构:如果它们太小，它们会失去结构，而如果太大，中心区域可能会坏死。因此，我们使用小鼠幼崽的腰部区域来生成具有适当大小的切片，以实现最佳的长期培养，但原则上，可以考虑其他部分。此外，我们选择使用腰部区域，因为腹侧和背侧区域彼此之间更容易区分。此外，该区域呈现出运动神经元和灰质比例较高的组织区域，这些是 SCI 中细胞替代疗法的感兴趣位点。 关于将细胞移植到切片中，主要问题与玻璃微针尖端的破裂有关。如果细胞传入的孔太大，则在显微注射过程中可能会对SC组织造成损伤。如果它太小，细胞堆积会阻塞针头，从而阻碍移植过程。移植程序应在 1 小时内完成，以尽量减少细胞痛苦和死亡。
所提出的方案为不同类型的调查提供了一种最佳和通用的工具。在这里，我们应用我们的长期平台来验证 h-SC-NES 细胞在小鼠 SC 组织内分化的第一阶段移植 30 天。所提方法的主要新颖性是共培养方案的优化。GM 的成分维持 SC 切片和移植的 h-SC-NES 细胞的长期神经元存活。事实上，转基因作为一种无血清培养基，与以前用于器官型切片培养的培养基相比，它维持了移植细胞向神经元命运的分化47。
关于所提出的SCI模型，通常在成年小鼠上进行实验。到目前为止，新生儿和成人 SC 之间最重要的差异与新生儿相对于成年小鼠发现的更高的再生潜力有关52。然而，这种差异对我们提出的协议类型没有影响，因为在这里我们关注的是移植细胞对宿主组织环境的反应，而不是对驻留神经元的再生能力的反应。SCI后新生小鼠和成年小鼠之间的另一个差异与成人中发生的神经胶质瘢痕的形成有关。所提出的模型没有考虑到这一方面，该模型没有考虑由原发性和继发性损伤引起的复杂生理病理过程。
关于应用，该平台还可用于研究移植细胞与SC器官型模型中存在的驻留电路之间的整合。基因工程工具已经在中枢神经系统中用于评估突触连接，并且可以在这方面加以利用53,54,55。特别是，可以通过评估移植细胞和 SC 离体组织之间突触的形成来研究和验证整合。这些长期器官型培养物也可用于检测神经保护剂和神经再生剂或新型分子/材料，或研究涉及 SC 的神经退行性疾病。为了研究特定的神经退行性疾病，该方案需要适应在病理学的相关阶段（即新生儿、青少年、成人）培养由相关模型生成的SC切片，例如携带特定病理学相关突变的转基因小鼠。总之，我们的方案和一般的器官型培养物，作为特定器官的外植体，具有弥合 2D 细胞培养物和体内模型之间差距的特征，证实了它们是基础研究和临床前测试的宝贵工具。

创伤性脊髓损伤 （SCI） 对患者和护理人员的身体、心理和经济后果1.已经进行了许多尝试，以不同的方法促进 SCI 中的轴突再生 2,3,4，并且通过细胞替代疗法在损伤部位的近端和远端神经元之间形成神经元中继，证明了一些有益的效果。人们对细胞疗法的兴趣仍在增长5，因为移植细胞可以发挥许多作用，包括提供营养支持、免疫调节、通过诱导可塑性再生丢失的神经回路、细胞替换和轴突髓鞘再生6。
最近，该领域的主要工作集中在人类神经干/祖细胞 （NSC/NPC） 上7。几项研究表明，NSCs/NPCs 调节星形胶质细胞反应8，促进促再生因子的分泌 9,10，并替换 SCI 中缺失的神经元细胞11,12。然而，支持移植细胞分化为功能性神经元的研究仍然很差。此外，移植细胞在损伤脊髓 （SC） 中的存活率和分化率低13，可能是因为移植细胞需要数周甚至数月才能在体内分化。此外，目前的研究尚未完全阐明细胞替代疗法的许多生化、分子、细胞和功能方面。在这种情况下，需要简单、快速且具有成本效益的模型来研究细胞植入的机制、移植细胞增殖、分化成特定类型或细胞亚群的能力，以及与驻留神经元形成突触的能力。
将组织学研究整合到电生理记录以及转录组和蛋白质组分析中，对于全面理解细胞移植后发生的分子级联反应是必要的。这无疑将加快临床前模型和临床研究中新细胞替代疗法的设计和验证。事实上，迄今为止，使用啮齿动物、大型动物和非人灵长类动物对于阐明移植后的许多细胞过程是值得的14.然而，由于成本高、伦理影响大以及生物体的复杂性，它们的使用往往不直接或不足以揭示生化和分子过程。此外，它们可能存在许多与生物学差异相关的缺点，包括种间（代谢）和种内变异性（性别、年龄）。 这些因素，加上压力情况等外部因素，可能会改变实验的结果及其在对人类的治疗转化方面的可预测性15,16,17。
因此，除了动物模型外，许多研究小组还采用2D体外细胞培养和离体器官型切片（离体培养）。2D细胞培养是在单细胞和/或细胞群水平上研究特定生物过程的最常用系统。然而，单层细胞培养并不能反映在整个生物体中发现的复杂性。缺乏组织结构和生理环境使得 2D 培养系统无法完全模拟所研究组织的关键结构、形态和功能方面 18,19,20。器官型培养可以克服其中的一些问题。器官型模型基于移植组织或器官的片段并将其在有限的时间内保持在体外21,22。特别是，外植组织的切片以精确的厚度生成，使营养物质能够轻松到达切片中的几乎所有细胞。它们可以从中枢神经系统的各个区域产生，例如海马体、下丘脑、小脑、丘脑、大脑皮层、黑质和纹状体以及脊髓23。器官型培养保留了组织结构、细胞的空间分布、细胞多样性和起源器官的环境（即细胞外基质组成）。此外，它们保留了原始的神经活动，细胞之间的连接，特别是外植体后的短距离回路。
与单层培养物和动物模型相比，这些方面为离体培养物提供了一些优势。它们保留了体内发现的关键组织特征，但降低了成本，并有可能进行不同类型的分子、细胞和功能实验，并准确调节培养环境参数 24,25,26,27,28,29.器官型切片也可用于通过类似于特定疾病的关键组织病理学特征来开发不同神经系统疾病的模型30。此外，原始多细胞组织环境的保留使它们成为药物筛选和测试神经保护和神经再生分子和材料的合适平台。
在这项工作中，我们建议使用 SC 器官型培养物作为模型来优化 NSC 移植。这并非微不足道，因为需要最佳的培养条件来保证宿主（SC组织）和移植物（NSC）数周的存活。不同的研究小组移植到器官型培养物中，脑源性和SC衍生的各种类型的细胞。大多数工作展示了间充质干细胞31,32,33，嗅鞘细胞34或NSCs的移植35,36,37,38,39,40，并评估了移植细胞与宿主细胞的相互作用，整个系统的存活率，以及移植的细胞是否分化为神经元或神经元样细胞在离体组织环境中 32,33,41。他们中的一些人评估了移植后细胞的再生潜力，观察它们在组织内的轴突生长37,40,41，少突胶质细胞移植前体的髓鞘化能力42，移植细胞向宿主组织的迁移43，以及移植细胞是否释放了推动促再生环境的因子31.目前研究的一个局限性是它们没有长期探索植入。
考虑到 NSC 似乎需要数周时间才能在体内分化 44,45，本研究的重点是如何生成和维持长期（≥30 天）小鼠 SC 切片长达 90 天。研究发现，切片保留了其原始的解剖结构，并随着时间的推移保持低而稳定的凋亡率和高细胞活力。我们观察到神经元标记物 RNA 结合 fox-1 同源物 3 （RBFOX3） 和神经丝轻链 （NFL） 的弥漫表达，随着时间的推移，后者在切片周围轴突发芽呈增加趋势，证明了它们的健康状况。此外，在神经元分化的第一阶段，我们成功地将表达GFP的人SC衍生的神经上皮干（h-SC-NES）移植到SC切片中。移植后维持 NSC 移植物 30 天，细胞在培养的所有期间均显示 GFP 表达。还发现移植后第二天 （DPT） 30 的细胞凋亡率与相同细胞40 中在 DPT 7 处观察到的凋亡率值一致。 细胞似乎植入组织环境并存活长达数周。
总之，我们的数据表明，可以在培养物中将 SC 器官型切片维持 3 个月，而不会影响其原始细胞结构和组织环境。最重要的是，它们可以用于在进行 体内 实验之前测试细胞疗法，从而降低成本和实验时间。在这里，我们详细说明了产生小鼠SC器官型切片的所有段落以及如何长期（≥30天）维持它们。此外，我们还深入解释了如何将鼻咽癌移植到切片中，以及如何保持鼻咽癌以进行下游分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-cleaved Caspase-3,&#160; (Asp175) (5A1E) (Rabbit)</td>
			<td>Cell Signaling Technology</td>
			<td>9661S</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>anti-GFP (Mouse) - monoclonal</td>
			<td>Sigma/Merck</td>
			<td>G6539</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>anti-Human Nuclei (Mouse) - monoclonal, clone 235-1&#160;</td>
			<td>Sigma/Merck</td>
			<td>MAB1281</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>anti-Human Nuclei (Rabbit)</td>
			<td>NeoBiotechnologies</td>
			<td>RBM5-346-P1</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>anti-NeuN (RBFOX3) (Rabbit) - polyclonal</td>
			<td>Sigma/Merck</td>
			<td>ABN78</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>anti-NFL (Mouse)</td>
			<td>Sigma/Merck</td>
			<td>MAB1615</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>anti-NFL H-Phospho (Rabbit) -polyclonal</td>
			<td>Biologend</td>
			<td>840801</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Aqua Polymount</td>
			<td>Poly-sciences</td>
			<td>18606-20</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Gibco</td>
			<td>PHC7074</td>
			<td></td>
		</tr>
		<tr>
			<td>Blades</td>
			<td>Leica</td>
			<td>118364227</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture graded water</td>
			<td>Sigma/Merck</td>
			<td>W3500-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from rat tail</td>
			<td>Sigma/Merck</td>
			<td>C7661</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope - A1 Confocal Microscope (Eclipse Ti)</td>
			<td>Nikon&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>Sigma/Merck</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Forceps</td>
			<td>World Precision Instruments</td>
			<td>15915</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>31330</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Sigma/Merck</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma/Merck</td>
			<td>gf144</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-2</td>
			<td>Stemgent</td>
			<td>03-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Sigma/Merck</td>
			<td>SRP3200</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries, 3.5&#34;&#160;</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti Mouse IgG Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti Mouse IgG Alexa Fluor 647</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21236</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Goat-anti Rabbit IgG Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11011</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Goat-anti Rabbit IgG Alexa Fluor 647</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21244</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Graph Pad-Prism</td>
			<td>Dotmatics</td>
			<td></td>
			<td>Software for Statistical Analysis</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14025-050</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma/Merck</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma/Merck</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentiviral prep</td>
			<td>Addgene</td>
			<td>17446-LV</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity assay kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>L32250</td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Gibco</td>
			<td>11090-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>to load cells in the needle for transplantation</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>VWR</td>
			<td>631-0909</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell cell culture membrane</td>
			<td>Sigma/Merck</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Miscroscope cover glasses</td>
			<td>VWR&#160;</td>
			<td>ECN 631-1572</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (35mm)</td>
			<td>VWR</td>
			<td>734-2317</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma/Merck</td>
			<td>P6148-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic pasteur pipette</td>
			<td>Sarstedt</td>
			<td>86.1171.010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic PicoPump</td>
			<td>World Precision Instruments</td>
			<td>PV830</td>
			<td>Microinjector for transplantation</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma/Merck</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade No 10 Sterile Stainless Steel</td>
			<td>VWR International</td>
			<td>SWAN3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle #3</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors</td>
			<td>World Precision Instruments</td>
			<td>501235</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope for imaging and acquisition</td>
			<td>Nikon&#160;</td>
			<td>SMZ18</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope for surgery</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers-Dumont #5-inox</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors, 8.5 cm</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical micropipette puller</td>
			<td>Shutter Instrument</td>
			<td>P-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>R&#38;D Systems</td>
			<td>1254/50</td>
			<td></td>
		</tr>
	</tbody>
</table>

long-term mouse spinal cord organotypic slice culture as a platform for validating cell transplantation in spinal cord injury

由于病理生理学复杂的脊髓损伤 （SCIs） 方法仍然缺乏。最有前途的再生方法之一是基于干细胞移植，以替换丢失的组织并促进功能恢复。在进行更昂贵和耗时的动物试验之前，应 在体外 和 体外 更好地探索这种方法的安全性和有效性。在这项工作中，我们展示了建立一个基于小鼠脊髓 （SC） 器官型切片移植人类神经干细胞的长期平台，以测试 SCI 的细胞替代疗法。
标准 SC 器官型培养物 在体外维持约 2 或 3 周。在这里，我们描述了一种优化的协议，用于长达 90 天的长期维护（≥30 天）。用于长期培养 SC 切片的培养基也针对将神经干细胞移植到器官型模型中进行了优化。将携带绿色荧光蛋白 （GFP） 报告基因的人 SC 衍生神经上皮干 （h-SC-NES） 细胞移植到小鼠 SC 切片中。移植后30天，细胞仍显示GFP表达和低凋亡率，表明优化的环境维持了它们在组织内的存活和整合。该协议代表了在SC组织中有效测试细胞替代疗法的有力参考。该平台将使研究人员能够对不同的细胞移植疗法进行 体外 预筛选，帮助他们在进行 体内 实验之前选择最合适的策略。

所描述的方法允许在P3阶段建立小鼠的SC器官型切片，并在健康条件下将其长时间维持在培养物中。此外，我们还展示了将细胞移植到切片中并共同培养它们长达 30 天的方案（图 1）。首先，我们展示了培养条件的优化和适用于用移植细胞长时间培养SC切片的方案（图2A）。在OM中，从DEV 0到DEV 2生成并维持切片，OM最初被提议作为维持SC切片47的最佳介质。然而，由于血清蛋白的存在，该培养基可能无法维持移植的神经前体细胞的神经元分化和成熟。事实上，在 DEV 3 中，我们测试了从 OM 到 GM 的转换，这是一种含有 Neurobasal 加 B27 的配方，它支持神经存活，并且不含血清，它会抑制正确的神经元分化，反而促进神经胶质命运48,49。
图2B显示了将DEV 3的培养基从OM切换到GM所获得的结果，与未接收到开关的SC切片（对照切片在OM中培养）相比。我们使用切片内NFL信号的分布作为神经元完整性的标志（图2B，C）。DEV 7 的切片在两种培养条件下都是健康的，显示出它们内部神经丝（NFL，绿色）的弥散分布。在 DEV 10 中，与在 OM 中培养的对照切片相比，在 GM 中培养的切片似乎更健康，正如 NFL 染色分布所记录的那样。我们还估计了图2B的代表性图像中显示的切片的NFL+面积（% NFL+面积/DAPI+面积）。估计的 NFL+ 面积在图 2C 的直方图中表示，证实了在两种条件下，NFL 信号在 DEV 7 处的切片中扩散分布。然而，在 DEV 10 时，对于 OM 培养条件，NFL 染色覆盖的估计面积会减少。
这些数据表明，在 DEV 3 时切换到 GM 对于 SC 切片的长时间培养 （DEV 10） 具有良好的耐受性。下一步，我们在更长的时间点测试了 GM:DEV 30、DEV 60 和 DEV 90。如图 3A，B 所示，切片在培养物中保持健康，直到 DEV 90。在每个时间点的切片中都发现 NFL 染色广泛存在，从中心区域出发的神经突切片周围有弥漫性发芽。事实上，我们估计了图3A所示切片的NFL+面积，并且随着时间的推移而增加，如图3B的直方图所示。我们还观察到神经元标记物RBFOX3的阳性，为切片的神经元分化提供了另一条证据。在每个时间点，我们还通过评估不同切片中对aCASP3阳性的细胞数量来检查凋亡率（图4A，B）。分析按照协议第 10.2 节中的描述进行。发现每个时间点的凋亡率（% aCASP3+ 细胞/DAPI+ 细胞总数）非常低（DEV 30、60 和 90 ± 分别为 0.85 ± 0.52%、0.71 0.27%、0.66 ± 0.45%），三个考虑的时间点之间没有显着差异（p 值 > 0.05，图 4B）。这些数据表明，与 aCASP3 相关的凋亡率在一段时间内保持稳定，并且与切片中 NFL 的广泛分布（图 4A）一起证实了切片在每个时间点的存活。
为了支持之前的数据，我们还进行了活/死测定，以评估切片在三个不同时间点的活力。我们使用钙黄绿素（绿色染色）来标记活细胞和代谢活性细胞，并使用 Sytox（青色染色）来评估细胞死亡。 如图 4C 中的直方图所示，从 DEV 30 到 DEV 90，代谢活性细胞的百分比略有增加（DEV 30、60 和 90 分别为 93.17 ± 5.21%、96.43 ± 3.02%、96.33 ± 3.10%），在最后两个时间点之间保持稳定（DEV 30 vs DEV 60 p 值 = 0.018;DEV 30 与 DEV 90 p 值 = 0.027;DEV 60 与 DEV 90 p 值 = 0.99）。我们发现随着时间的推移，细胞死亡水平较低（DEV 30、60 和 90 分别为 6.83 ± 5.21%、3.57 ± 3.02%、3.66 ± 3.10%），并且发现 DEV 30 和以后的时间点 DEV 60 和 DEV 90 之间存在显着差异（DEV 30 vs DEV 60 p 值 = 0.018;DEV 30 与 DEV 90 p 值 = 0.027;DEV 60 与 DEV 90 p 值 = 0.99）。这些数据与细胞凋亡率相关联，证实了切片随时间推移的存活率，并支持所执行的长期培养方案的有效性。
一旦确定了 SC 切片长时间培养的可行性，我们就通过在神经元分化的第一阶段移植 h-SC-NES 细胞来挑战该系统。我们测试了 h-SC-NES 细胞，因为它们在 SCI 治疗12 中显示出有希望的结果。将h-SC-NES细胞移植到小鼠SC切片中的程序在方案第6节中描述。SC 切片和移植的 h-SC-NES 细胞维持至 DPT 30。在分化的 DIV 10（神经前体阶段）将细胞移植到 DEV 4 器官型切片中，如图 5A 的方案方案所示。监测移植细胞在培养物中 GFP 的表达长达 30 天。 图5B 显示了在不同DPT下移植GFP+ 细胞的SC切片的代表性实时图像。GFP随时间推移的稳定表达（图5B 和 图6A）表明，在先前优化的培养条件下，细胞存活到SC组织中。我们还检查了移植细胞的凋亡率，如方案第10.2节所述。发现30 DPT后凋亡率（% aCASP3 + 细胞/胡-Nu + 细胞总数）非常低（0.44±0.34%）（图6B）。此外，发现 DPT 30 时的凋亡率与 DPT 7 时相同类型细胞的凋亡率一致，如先前报道的40 所示，表明培养物随着时间的推移而稳定。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66704/66704fig01.jpg"> 图 1:协议的工作流程。 代表性方案，显示了所执行协议的一般工作流程。（A） 左图，总结了小鼠幼崽在 P3 处分离的 SC 产生小鼠 SC 切片和 SC 器官型切片的长期培养的方案。（B） 右边是总结将表达 GFP 的 h-SC-NES 细胞移植到小鼠 SC 器官型切片中的方案。移植细胞在移植后维持 30 天。缩写:h-SC-NES=人脊髓来源的神经上皮茎;GFP = 绿色荧光蛋白;DEV = 离体日;DPT = 移植后第二天;NFL = 神经丝轻链;RBFOX3= RNA 结合 fox-1 同源物 3;aCASP3 = 活性 Caspase-3;SC=脊髓。 <a href="https://www.jove.com/files/ftp_upload/66704/66704fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66704/66704fig02v2.jpg"> 图2:长期培养条件的优化。 （A） 测试OM和GM的协议的代表性方案。 对照组的OM维持至DEV 7-10。在DEV 3时将培养基切换到GM以处理过的切片;然后，将它们固定在 DEV 7-10 上，以便与对照进行比较。（B） 比较在不同条件下培养的 DEV 7 和 10 的小鼠 SC 器官型切片的代表性图像。对切片进行细胞骨架标记物神经丝（NFL，绿色）染色。用转基因培养的切片中 NFL 染色的广泛分布表明总体存活率和分化。细胞核用DAPI复染。比例尺 = 500 μm。 （C） 图 1B 所示切片中 NFL 覆盖面积估计值的代表性直方图。在 DEV 10 时，在 OM 培养条件下，NFL 表面积减小。缩写:DEV = day ex vivo;DAPI = 4'，6-二脒基-2-苯基吲哚;NFL = 神经丝轻链。;OM = 器官型培养基;GM = 移植培养基;SC = 脊髓。 <a href="https://www.jove.com/files/ftp_upload/66704/66704fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66704/66704fig03v3.jpg"> 图 3:长期培养的小鼠 SC 器官型切片。 （A） 切片在培养物中保持至 DEV 90。免疫荧光测定显示细胞骨架标记物神经丝（NFL，绿色）和核神经元标记物 RBFOX3（红色）分布广泛，证明了它们在长期培养后的健康状况和神经元身份。值得注意的是，随着时间的推移，NFL+ 轴突会在切片周围弥漫性地发芽。细胞核用DAPI复染。比例尺 = 500 μm。 （B） 图 A 中显示的 NFL+ 面积和时间估计值以及 RBFOX3+ 面积估计值的代表性直方图 。 NFL+ 神经突面积随着时间的推移而增加。缩写:DEV = day ex vivo;DAPI = 4'，6-二脒基-2-苯基吲哚;NFL = 神经丝轻链;SC = 脊髓;RBFOX3= RNA 结合 fox-1 同源物 3。 <a href="https://www.jove.com/files/ftp_upload/66704/66704fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66704/66704fig04.jpg"> 图 4:SC 切片中细胞活力随时间的评估。 （A） DEV 60 处的 aCASP3（红色）和 NFL（绿色）染色的器官型切片的代表性图像。比例尺 = 100 μm。 NFL 显示漫反射图案。稀有细胞的凋亡标志物 aCASP3 呈阳性（插图:1-2-3）。（B）不同时间点切片凋亡率分析。平均 ± SD，N（重复）= 6 个切片，n（总细胞数）>每个切片 1,000，Kruskal-Wallis 检验，多重比较，p 值 > 0.05。凋亡率随时间推移保持稳定。在图 A 的插图 1-2-3 中，可以观察到 aCASP3 阳性细胞的细节（红色染色，白色箭头）。小红点标记细胞碎片和脓结核。比例尺 = 50 μm。 （C） 在 DEV 90 时对 SC 切片进行的活/死测定的代表性图像:代谢活性细胞用钙黄绿素标记为绿色，而死亡和受损细胞用 Sytox 标记浅蓝色（青色）。两个直方图显示了钙黄绿素（左侧）和 Sytox（右侧）阳性的细胞占细胞总数的百分比。对于均值± SD，N（重复）= 6 个切片，n（总细胞数）>每个切片 1,000，Kruskal-Wallis 检验，多重比较， DEV 30 vs DEV 60 p 值 = 0.018;DEV 30 与 DEV 90 p 值 = 0.027;DEV 60 与 DEV 90 p 值 > 0.99。缩写:DEV = day ex vivo;DAPI = 4'，6-二脒基-2-苯基吲哚;NFL = 神经丝轻链;SC = 脊髓;aCASP3 = 活性半胱天冬酶-3。 <a href="https://www.jove.com/files/ftp_upload/66704/66704fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66704/66704fig05.jpg"> 图 5:将 h-SC-NES 细胞移植到小鼠器官型切片中。 （A） 移植方案的代表性方案。细胞在分化的 DIV 10 时作为神经前体移植到 DEV 4 器官型切片中。（B） 移植用表达 GFP 的 h-SC-NES 细胞移植的小鼠器官型切片的代表性图像，直至 DPT 30。用携带 GFP 基因的慢病毒载体转导细胞。GFP随时间推移的表达证实了它们的活力和对切片环境的适应性。比例尺 = 500 μm。缩写:DIV = 分化前的第一天;h-SC-NES = 人脊髓来源的神经上皮茎;GFP = 绿色荧光蛋白;DEV = 离体日;OM = 器官型培养基;GM = 移植培养基;DPT = 移植后天数。 <a href="https://www.jove.com/files/ftp_upload/66704/66704fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66704/66704fig06.jpg"> 图 6:移植后 30 天后移植的 h-SC-NES 细胞的凋亡率评估。 （A） 移植有表达 GFP 的 h-SC-NES 细胞的小鼠器官型切片的代表性图像。用携带 GFP 基因的慢病毒载体转导细胞，以便在移植后监测它们进入切片。GFP随时间推移的表达证实了它们的活力和对切片环境的适应性。显示的时间点为 DPT 30;对细胞进行人核（青色）和 aCASP3（红色）染色。比例尺 = 150 μm。 （B） 左侧是 DPT 30 时移植成片的细胞凋亡分析的代表性饼图（N（重复）= 5 个切片，n （细胞） = 5,000），右侧是 胡-Nu+ 细胞的插图和对 aCASP3 呈阳性的细胞的详细信息（白色箭头）。比例尺 = 75 μm。小红点标记细胞碎片和脓结核。缩写:h-SC-NES=人脊髓来源的神经上皮茎;GFP = 绿色荧光蛋白;DPT = 移植后第二天;DAPI = 4'，6-二脒基-2-苯基吲哚;NFL = 神经丝轻链;aCASP3 = 活性半胱天冬酶-3;胡-努 = 人类细胞核。 <a href="https://www.jove.com/files/ftp_upload/66704/66704fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
表1:本协议中使用的溶液组成。<a href="https://www.jove.com/files/ftp_upload/66704/66704_Tables%20of%20Solutions%20rev.xlsx" target="_blank">请按此下载此表格。</a>

Read this Scientific Article Protocol about Author Spotlight: Long-Term Spinal Cord Slice Culture for Advancing Spinal Cord Regeneration Therapies at JoVE.com

作者聚焦:用于推进脊髓再生疗法的长期脊髓切片培养

在本文中，我们提供了一种可重复的方法来生成和维持移植神经干细胞的长期脊髓器官型切片，作为测试细胞替代疗法的 离体 模型。

长期小鼠脊髓器官型切片培养作为验证脊髓损伤细胞移植的平台

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches ...

我们有兴趣开发一种有前途的再生方法来解决脊髓损伤。在本文中，我们验证了脊髓器官型模型在脊髓研究中测试细胞替代疗法。到目前为止，脊髓器官型模型在体外培养物中维持两到三周。而传代培养基对于神经干细胞植入、分化和成熟来说是次优的。细胞替代疗法仍然需要改进，以宣布移植细胞重建丢失的电路的能力。通过该协议，我们提供了一个新颖的、长期的离体平台，以解决细胞移植相关问题，如移植神经干细胞的存活、整合和成熟率。该平台将有助于研究人员找到细胞移植的最佳策略，减少体内验证所需的动物数量。我们的方案简单、快速且经济高效，可用于执行概念验证和优化研究。

long-term-mouse-spinal-cord-organotypic-slice-culture-as-platform-for

Research

JoVE Journal

Neuroscience

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury

1.4K 次观看.  University of Pisa. 我们有兴趣开发一种有前途的再生方法来解决脊髓损伤。在本文中，我们验证了脊髓器官型模型在脊髓研究中测试细胞替代疗法。到目前为止，脊髓器官型模型在体外培养物中维持两到三周。而传代培养基对于神经干细胞植入、分化和成熟来说是次优的。细胞替代疗法仍然需要改进，以宣布移植细胞重建丢失的电路的能力。通过该协议，我们提供了一个新颖的、长期的?...

视频: 作者聚焦:用于推进脊髓再生疗法的长期脊髓切片培养

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

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 ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

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 ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

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 ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

脊髓损伤 wistar 大鼠跑步机的作业训练

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

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, ...