这项工作得到了国家科学技术委员会（CONACYT）（702361）授予的奖学金的支持。作者承认博士国立理工学院国立生物科学学院生物化学科学课程。

该研究是根据墨西哥官方标准NOM-062-ZOO-1999和实验动物护理和使用指南进行的。该研究获得了墨西哥儿童医院研究、伦理和生物安全委员会 （HIM/2023/006） 和墨西哥总医院研究和生物伦理委员会 Eduardo Liceaga 的批准 （DI/21/501/04/62）。从墨西哥儿童医院获得三只 6 至 8 周龄的 C57BL/6 小鼠，在隔离条件下在通风架中饲养，符合机构动物护理和使用指南。
1.材料和试剂的制备
<ol><li>将不含Ca2+ 和Mg2+ 的PBS以及Hank平衡盐溶液（HBSS）预热至37°C。</li>
	<li>用 100 μg/mL 的 Liberase 和 4 μg/mL 的 DNase （HBSS-LD） 补充 HBSS 溶液。确保溶液的生理pH值保持在7.4。 注意：每条脊髓需要 1 mL 的工作溶液。</li>
	<li>准备用于密度梯度离心的等渗90%溶液（市售，见 材料表）并将其预热至37°C。</li>
	<li>通过将5%热灭活胎牛血清（FBS）与PBS混合来制备染色缓冲液，并将混合物储存在冰上。</li>
	<li>将Dulbecco改良的Eagle's中高葡萄糖（1g / L）（DMEM）预热至37°C。</li>
	<li>用 10% FBS、2 mM L-谷氨酰胺、200 U/mL 青霉素、200 μg/mL 链霉素和 500 pg/mL 两性霉素 B（参见 材料表）补充 DMEM 培养基以制备 DMEM-S。</li>
</ol>2. 脊髓的解剖和准备
<ol><li>用腹膜内过量的戊巴比妥以150mg / kg的剂量对小鼠实施安乐死。使用70%乙醇对小鼠的胸部和背部进行消毒。 注意：在进行小鼠解剖之前，请确认安乐死成功。</li>
	<li>剃掉胸腰椎区域，并用胶带将四肢固定在解剖板上。</li>
	<li>使用手术刀，沿着从枕骨到骶骨区域的背中线小心切开（图2）。</li>
	<li>在立体手术显微镜的帮助下，分层解剖，直到到达腰椎并识别最后一个肋弓。 注意：13 个肋弓在胸椎中铰接;解剖时，它们变得比 L1 椎骨2 更上可见。</li>
	<li>确定最后一个颈椎和骶骨，然后切开脊柱（图2）。</li>
	<li>利用解剖钳，进行椎骨背侧椎板切除术以提取脊髓（图3A-D）。 注：除前两个颈椎（寰椎和轴椎）外，所有可移动椎骨在各个区域（颈椎、胸椎或腰椎）都具有共同的形态设计。每个典型的可活动椎骨在其前端都有一个圆柱形椎体。附着在身体后部的是称为椎弓（神经弓）的骨弓，由椎板和棘突组成。在这些结构之间是椎孔15。</li>
	<li>切除通过孔（背根和腹根）出现的神经，这些神经构成周围神经系统，可能含有浸润的巨噬细胞（图3D）。 注意：没有必要去除软脑膜或脑膜，因为密度梯度培养基可有效消除污染的星形胶质细胞。此外，保留脑膜可缩短纯化时间并增强活力。</li>
</ol>3.小胶质细胞的组织消化和分离
<ol><li>将脊髓放在解剖板上，使用 Vannas 剪刀将脊髓切成 2 毫米的小块，平均产生 10 块。</li>
	<li>将脊髓切片转移到2mL平底微管中（图3E，F）。</li>
	<li>加入 1 mL HBSS-LD 工作溶液（步骤 1.2）。在37°C孵育25分钟，每5分钟剧烈涡旋一次。</li>
	<li>将每个消化的内容物通过 40 μm 过滤器，然后加入 7 mL 含有 2% FBS 的 HBSS 以中和消化。</li>
	<li>使用 1 mL 注射器柱塞粉碎剩余的组织。将悬浮液在50mL锥形管中以220× g，4°C离心5分钟。</li>
	<li>使用 1 mL 微量移液管弃去上清液，并将沉淀重悬于 15 mL 锥形管中含有 2% FBS 的 2 mL HBSS 中。与 2 mL 90% 等渗密度梯度培养基混合。在160×g，18°C下离心25分钟。 注意： 利用缓慢加速并避免使用制动器。</li>
	<li>使用移液器取回接口并将其转移到 15 mL 锥形管中。用10mL PBS洗涤细胞，并在220× g，4°C下离心5分钟。</li>
	<li>将细胞重悬于DMEM-S（步骤1.6）中，并将它们储存在冰上。</li>
</ol>4. 确定纯度/活力
<ol><li>为了使用血细胞计数器室定量活细胞，使用0.4%台盼蓝染色剂（参见 材料表）。 注：平均而言，一只小鼠提供4-600万个细胞。</li>
	<li>要 通过 流式细胞仪定量活细胞，请使用市售的胺反应性荧光染料染色（参见 材料表）。<ol><li>将 100 万个细胞转移到 5 mL 聚苯乙烯圆底 （FACS） 管中。用PBS以1：1000的比例稀释荧光染料（产生活性工作溶液）。</li>
		<li>将样品与活力溶液在4°C的黑暗中孵育30分钟。用冰冷的PBS洗涤细胞两次。将细胞重悬于400μL染色缓冲液中（步骤1.4）。</li>
		<li>在4°C下用2.4G2抗FcRIII / I（参见 材料表）在冰冷的染色缓冲液中封闭细胞15分钟。</li>
		<li>通过将荧光标记的单克隆抗体 CD45-PerCP 和 CD11b-eFluor 450 加入染色缓冲液中（稀释度为 1：300）（参见 材料表）来配制抗体染色混合物。 注意：虽然这两种技术都可用，但请选择最符合后续协议的技术。</li>
		<li>将样品与抗体染色混合物在4°C的黑暗中孵育30分钟。用冰冷的染色缓冲液洗涤细胞两次。将细胞重悬于 400 μL 染色缓冲液中。</li>
		<li>在流式细胞仪上采集 250,000 个事件，如步骤 6 中的设门策略所述（图 4）。</li>
	</ol></li>
</ol>5.免疫荧光染色
<ol><li>将预处理的盖玻片放入 6 孔板的孔中。 注：要处理盖玻片，将它们置于含有500μL-聚赖氨酸（0.01mg / mL，参见 材料表）溶液的培养皿中，并孵育1小时。然后，用蒸馏水清洗三次，然后晾干。</li>
	<li>使用 500 μL DMEM-S 溶液将 200,000 个细胞接种到处理过的盖玻片上。孵育24小时。</li>
	<li>取出盖玻片，在室温下使用3.7%多聚甲醛（PFA）固定细胞20分钟。用冰冷的PBS清洗盖玻片三次。</li>
	<li>在室温下用0.2%Triton X-100在PBS中透化细胞15分钟。随后，用冷PBS洗涤盖玻片三次。在室温下用0.2%BSA在PBS中封闭细胞1小时。</li>
	<li>在封闭溶液中稀释荧光标记的单克隆抗体（以1：300稀释度，参见 材料表），并在室温下孵育1小时。用冰冷的PBS清洗盖玻片三次。</li>
	<li>将含有DAPI的封固剂涂在载玻片上，然后用盖玻片密封。 注意：载玻片可以立即使用荧光或共聚焦显微镜进行分析，也可以在 -20°C 下储存长达 3 个月。</li>
</ol>6. 脊髓小胶质细胞的门控策略
<ol><li>生成点图并建立门以基于活力染色和空荧光通道16 分离活细胞。排除非活细胞。</li>
	<li>利用前向和侧向散射参数生成另一个图以消除碎片16。</li>
	<li>开发额外的点图并分析 CD11b 和 CD45 的表达以区分小胶质细胞。</li>
</ol>

小鼠脊髓中小胶质细胞的提取和酶促纯化

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作者声明没有利益冲突。

由于小胶质细胞在大脑稳态中的重要性，已经开发了许多用于研究小胶质细胞的方案。在这些方法中，小胶质细胞通常来源于胚胎或新生大鼠和小鼠的大脑半球17。有限数量的研究涉及从成年小鼠脊髓中纯化小胶质细胞13,14。这些技术包括使用胶原酶和/或木瓜蛋白酶以及DNAse进行酶消化，通常与使用Percoll或OptiPrep的梯度分离相结合。然?...

脊椎动物的定义特征是脊柱或脊柱，其中脊索已被一系列称为椎骨的分段骨骼所取代，这些骨骼被椎间盘分割。这种骨质物质的连续性塑造了椎管，椎管是一个包围和保护脊髓的腔1。在啮齿动物属中，脊柱通常由7个颈椎、13个胸椎、6个腰椎和数量不等的尾椎2,3组成。脊髓的长度与脊柱的长度相似，末端丝是将脊髓固定在骶骨上的非神经结构。此外，神经纤维通过椎间孔1 排出。
哺乳动物中枢神经系统的发育和正常功能在很大程度上取决于神经系统常驻巨噬细胞（称为小胶质细胞4）的活动。尽管小胶质细胞最初被描述为大脑驻留吞噬细胞，但最近的研究将许多动态功能归因于这些细胞 5,6。小胶质细胞的大小在稳态中为 7 至 10 μm;它们被认为是体内用途最广泛的细胞之一，可以在形态和功能上适应不断变化的环境7.这些细胞在胚胎期和成体阶段都表现出高度异质性8,9，而在成体阶段，它们也表现出基于其时空背景的复杂功能异质性10。小胶质细胞的异质性和多种功能允许在脊髓和大脑中产生不同的基因表达和行为。研究表明，与大脑相比，CD11b、CD45、CD86 和 CCR9 在脊髓中的表达更高 8,9。
脑小胶质细胞分离存在多种方案11,12;然而，脊髓小胶质细胞只有少数存在13,14。优化从脊髓中纯化小胶质细胞的方法有助于开展多项专注于发现小胶质细胞生理学的研究。该协议旨在描述小鼠脊髓的简单且高度可重复的提取和小胶质细胞的纯化（图1）。

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	<tbody>
		<tr>
			<td>15 mL collection tubes</td>
			<td>Corning, USA</td>
			<td>430790</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microtubes</td>
			<td>Axygen, USA</td>
			<td>MCT-200-G</td>
			<td></td>
		</tr>
		<tr>
			<td>2.4G2 anti-FcR</td>
			<td>BioLegend, USA</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL collection tubes</td>
			<td>Corning, USA</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin b)</td>
			<td>Gibco, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody CD11b eFluor 450 anti-mouse</td>
			<td>eBioscience, USA</td>
			<td>48-0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody CD45 PerCP anti-mouse&#160;&#160;</td>
			<td>BioLegend, USA</td>
			<td>103130</td>
			<td></td>
		</tr>
		<tr>
			<td>Balanced salt solution (PBS) calcium- magnesium-free</td>
			<td>Corning, USA</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Cell Strainer 40 &#956;m</td>
			<td>Corning, USA</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6-well Clear Not Treated&#160;</td>
			<td>Corning, USA</td>
			<td>CLS3736</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Heating Shaking Drybath&#160;</td>
			<td>Thermo Scientific Digital HS Drybath, USA</td>
			<td>88870001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps for microsurgery</td>
			<td>FT by DUMONT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche, USA</td>
			<td>4536282001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle&#180;s Medium-high glucose (DMEM)&#160;</td>
			<td>Merck, USA</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tube</td>
			<td>Thermo, USA</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>PAN Biotech, Alemania</td>
			<td>P30-3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer Cytoflex&#160;</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s balanced salt solution&#160;</td>
			<td>Merck, USA</td>
			<td>H2387</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning, USA&#160;</td>
			<td>15393631</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TM&#160;</td>
			<td>Roche, USA</td>
			<td>5401119001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber Counting Chambers</td>
			<td>China</td>
			<td>1103</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll&#160;</td>
			<td>Merck, USA</td>
			<td>17089101</td>
			<td>density gradient centrifugation&#160;</td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution&#160;</td>
			<td>Merck, USA</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel No. 25&#160;</td>
			<td>HERGOM, Mexico</td>
			<td>H23</td>
			<td></td>
		</tr>
		<tr>
			<td>Snaplock Microcentrifuge Tubes 2 mL</td>
			<td>Axygen, USA</td>
			<td>10011-680</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic microscope</td>
			<td>Velab, Mexico</td>
			<td>HG927831</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight surgical scissors (10 cm)</td>
			<td>HERGOM, Mexico</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Vannas scissors</td>
			<td>HERGOM, Mexico</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>Merck, USA</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue Stain 0.4%&#160;</td>
			<td>Merck, USA</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>DLAB, China</td>
			<td>8031102000</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua Fixable Viability Kit</td>
			<td>BioLegend, USA</td>
			<td>423102</td>
			<td>amine-reactive fluorescent dye staining&#160;</td>
		</tr>
	</tbody>
</table>

rapid and efficient enrichment of mouse spinal cord microglia

脊柱定义了脊椎动物并塑造了椎管，椎管是一个包围和保护脊髓的腔体。哺乳动物中枢神经系统的正常发育和功能在很大程度上依赖于常驻巨噬细胞（称为小胶质细胞）的活性。小胶质细胞具有异质性和多功能性，可在脊髓和大脑内实现不同的基因表达和行为。许多研究探索了脑小胶质细胞的功能，并广泛地详细介绍了纯化方法。然而，小鼠脊髓中小胶质细胞的纯化缺乏全面的描述。相比之下，与未精制的提取物相比，使用高度纯化的胶原酶在中枢神经系统组织中缺乏报告。在这项研究中，从 8-10 周龄的 C57BL/6 小鼠身上切除脊柱和脊髓。随后的消化采用高度纯化的胶原酶，小胶质细胞纯化采用密度梯度。对细胞进行流式细胞术染色，通过 CD11b 和 CD45 染色评估活力和纯度。结果显示，平均存活率为80%，平均纯度为95%。总之，小鼠小胶质细胞的操作涉及用高度纯化的胶原酶消化，然后是密度梯度。这种方法有效地产生了大量的脊髓小胶质细胞群。

利用小鼠脊髓组织，使用高度富含胶原酶和热溶菌素的混合物进行酶消化。所得的消化组织通过40μm过滤器以去除未消化的物质。收集的细胞通过Percoll密度梯度富集，下部为90%，上部为45%。然后用CD45和CD11b抗体对界面内富含小胶质细胞的细胞进行染色，并进行流式细胞术分析（图1）。
从枕骨区域延伸到骶骨的脊柱解剖涉及椎旁肌肉组织的暴露和解剖。?...

Watch this Scientific Journal Video about Microglia Research on Spinal Cord Heterogeneity and Purification at JoVE.com

作者聚焦：小胶质细胞对脊髓异质性和纯化的研究 

小胶质细胞被认为是体内最通用的细胞之一，能够进行形态和功能适应。它们的异质性和多功能性能够维持大脑稳态，同时也与各种神经系统病理有关。在这里，描述了一种净化脊髓小胶质细胞的技术。

小鼠脊髓小胶质细胞的快速高效富集

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and ...

我们的研究重点是小胶质细胞的异质性，它可以在不同的病理和区域（如大脑和脊髓）中表现出促炎和抗炎行为。我们的目标是更深入地了解脊髓小胶质细胞在不同情况下的行为。小胶质细胞的异质性使得很难对它们进行分类。今年，提出了一种新的、广泛的分类，它超越了促炎或抗炎状态。目前，从神经病理学动物模型中分离脊髓小胶质细胞时实现高纯度和凝胶化是一项重大的实验和挑战。我们在慢性疼痛期间在脊髓小胶质细胞中发现了一种新的细胞因子信号通路。我们的方案允许更可控的体细胞消化，然后是密度梯度。与目前用于从脊髓、大脑或胚胎中纯化小胶质细胞的技术相比，两者都减少了星形胶质细胞的污染。

rapid-and-efficient-enrichment-of-mouse-spinal-cord-microglia

Microsoft Bing

Research

JoVE Journal

Neuroscience

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

1.5K 次观看.  Mexico Children's Hospital. 我们的研究重点是小胶质细胞的异质性，它可以在不同的病理和区域（如大脑和脊髓）中表现出促炎和抗炎行为。我们的目标是更深入地了解脊髓小胶质细胞在不同情况下的行为。小胶质细胞的异质性使得很难对它们进行分类。今年，提出了一种新的、广泛的分类，它超越了促炎或抗炎状态。目前，从神经病理学动物模型中分离脊髓小胶质细胞时实现高纯度和凝胶化是?...

视频: 作者聚焦：小胶质细胞对脊髓异质性和纯化的研究 

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

科研

神经科学

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 ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

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 ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

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 ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

小鼠轻度创伤性脑损伤的电磁控制闭头模型

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

从成年小鼠脊髓中高效提取星形胶质细胞和小胶质细胞

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

作者聚焦：从成年小鼠脊髓中分离神经胶质细胞 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...