Name: induction of leptomeningeal cells modification via intracisternal injection
Uploaded: 2020-05-07T14:30:00
Description: Watch this Scientific Journal Video about Induction of Leptomeningeal Cells Modification Via Intracisternal Injection at JoVE.com

这项研究得到了瑞典研究理事会、瑞典癌症协会、瑞典战略研究基金会、克努特·奥赫·爱丽丝·瓦伦贝格斯·斯蒂夫瑟斯和卡罗林斯卡研究所干细胞和再生医学战略研究方案的资助。

特此提出的外科手术得到了斯德哥尔摩诺拉·久尔夫·德尔塞茨卡·纳姆德的批准，并符合该研究所（瑞典卡罗林斯卡研究所）提供的规格。
注：宫内注射可以灵活适应多种研究目的。我们下面介绍了一个程序，用于根据携带R26R-tdTomato12和CreER的转基因小鼠生产线注射内毒素，有效地标记蛋白酶细胞的命运映射，后者在Cx30促进12剂13</...

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A., 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=Retinoic+acid+from+the+meninges+regulates+cortical+neuron+generation.">Retinoic acid from the meninges regulates cortical neuron generation.</a> Cell. 139 (3), 597-609 (2009).</li><li>Bifari, F., 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=Neurogenic+Radial+Glia-like+Cells+in+Meninges+Migrate+and+Differentiate+into+Functionally+Integrated+Neurons+in+the+Neonatal+Cortex.">Neurogenic Radial Glia-like Cells in Meninges Migrate and Differentiate into Functionally Integrated Neurons in the Neonatal Cortex.</a> Cell Stem Cell. 20 (3), 360-373 (2017).</li><li>De Bock, M., 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=A+new+angle+on+blood-CNS+interfaces:+a+role+for+connexins?.">A new angle on blood-CNS interfaces: a role for connexins?.</a> FEBS Letters. 588 (8), 1259-1270 (2014).</li><li>Ramos, M., 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=Cisterna+Magna+Injection+in+Rats+to+Study+Glymphatic+Function.">Cisterna Magna Injection in Rats to Study Glymphatic Function.</a> Methods in Molecular Biology. 1938, 97-104 (2019).</li><li>Xavier, A. L. 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=Cannula+Implantation+into+the+Cisterna+Magna+of+Rodents.">Cannula Implantation into the Cisterna Magna of Rodents.</a> Journal of Visualized Experiments. (135), (2018).</li><li>Iliff, J. J., 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=A+paravascular+pathway+facilitates+CSF+flow+through+the+brain+parenchyma+and+the+clearance+of+interstitial+solutes,+including+amyloid+beta.">A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta.</a> Science Translational Medicine. 4 (147), (2012).</li><li>Coureuil, M., Lecuyer, H., Bourdoulous, S., Nassif, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+journey+into+the+brain:+insight+into+how+bacterial+pathogens+cross+blood-brain+barriers.">A journey into the brain: insight into how bacterial pathogens cross blood-brain barriers.</a> Nature Reviews Microbiology. 15 (3), 149-159 (2017).</li><li>Madisen, 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=Transgenic+mice+for+intersectional+targeting+of+neural+sensors+and+effectors+with+high+specificity+and+performance.">Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance.</a> Neuron. 85 (5), 942-958 (2015).</li><li>Slezak, M., 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=Transgenic+mice+for+conditional+gene+manipulation+in+astroglial+cells.">Transgenic mice for conditional gene manipulation in astroglial cells.</a> Glia. 55 (15), 1565-1576 (2007).</li><li>Hardy, S. J., Christodoulides, M., Weller, R. O., Heckels, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+Neisseria+meningitidis+with+cells+of+the+human+meninges.">Interactions of Neisseria meningitidis with cells of the human meninges.</a> Molecular Microbiology. 36 (4), 817-829 (2000).</li><li>Colicchio, 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=The+meningococcal+ABC-Type+L-glutamate+transporter+GltT+is+necessary+for+the+development+of+experimental+meningitis+in+mice.">The meningococcal ABC-Type L-glutamate transporter GltT is necessary for the development of experimental meningitis in mice.</a> Infection and Immunity. 77 (9), 3578-3587 (2009).</li><li>Ricci, S., 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=Inhibition+of+matrix+metalloproteinases+attenuates+brain+damage+in+experimental+meningococcal+meningitis.">Inhibition of matrix metalloproteinases attenuates brain damage in experimental meningococcal meningitis.</a> BMC Infectious Diseases. 14, 726 (2014).</li></ol>

此处概述的协议提供了一个简单且可重复的过程，用于标记脂肪映射的利普托明尼细胞。我们使用甲氧西芬（塔莫西芬的活性代谢物）的内腔注射，诱导Cx30-CreER中tdTomato荧光报告器的表达;R26R-tdTomato小鼠1212，1313。
与其他用于通过气囊9获得脑脊液的规程相比，我们的方法确保了安全手动管理，这要归功于使用弯曲?...

脑皮细胞是一种类似成纤维细胞的细胞群，它们组织成一层薄薄的细胞，覆盖大脑，表达与胶原蛋白交联有关的基因（例如，Dcn和Lum），以及建立脑脑电明屏障（例如，Cldn11）1、2。1,2脑膜细胞涉及广泛的生理功能，从严格控制脑脊液排水3到指导发育中的大脑中神经祖细胞44、5。5最近的一项研究还提出，新生儿中的耳皮层可能含有径向胶质状细胞，这些细胞会迁移到脑皮质瘤中，并发育成功能性皮质神经元6。
细胞体位于靠近表面星体细胞附近，并与它们共享，以及其他帕伦奇马尔星体，表达connexin-30（Cx30）7。7下面概述的外科手术允许通过一次性将内毒素输送到转基因小鼠的水箱中，在Cx30+ 细胞中表达tdTomato（即使用CreER-loxP系统进行命运映射）对这些脑膜炎细胞进行+广泛和具体的标签。Endoxifen 是塔莫西芬的活性代谢物，与塔莫西芬一样诱导克里尔表达细胞的重组。然而，它是推荐用于局部应用的解决方案，因为它溶解在5-10%的DMSO中，而不是高浓度的乙醇。此外，内氧西芬不会跨越脑脑阻隔，从而能够对脑皮质细胞进行特定的重组，而无需标记基础Cx30+天体胶质种群（参见代表性结果）。
此处介绍的技术旨在通过直接进入水仙子，手动安全地将化合物注入脑脊液中。与其他需要颅骨切除术的更具侵入性的程序不同，这种方法允许在不造成颅骨或脑筋皮损伤的情况下注入化合物。因此，它与由激活皮质胶质细胞触发的炎症反应的诱导无关。与,88、9、109之前描述的其他注射策略类似，本办法依赖于覆盖水箱的肌酸-四面体膜的外科暴露，在钝刻颈部肌肉解剖后。10然而，与其他程序不同，我们建议在尖端弯曲针头，在给剂期间，针对骨骼可以稳定。这将防止针头穿透太深和损害潜在的小脑和髓线的风险。
此外科手术与谱系追踪调查兼容，旨在绘制细胞身份的变化和通过层形层的迁移路线。它也可以适应基因消融研究，旨在探讨利皮质细胞在健康和疾病中的作用，如它们对皮质发育5或细菌性脑膜炎的传播的贡献,3，11。最后，当与野生动物中荧光示踪剂的传递相结合时，可用于跟踪脑脊液运动。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia unit</td>
			<td>Univentor 410</td>
			<td>8323102</td>
			<td>Complete of vaporizer, chamber, and tubing that connects to chamber and mouse head holder</td>
		</tr>
		<tr>
			<td>Anesthesia (Isoflurane)</td>
			<td>Baxter Medical AB</td>
			<td>000890</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Sigma-Aldrich</td>
			<td>PVP1</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Orion Pharma AB</td>
			<td>014920</td>
			<td>Commercial name Rymadil</td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>Carl Roth</td>
			<td>0258.1</td>
			<td>Use silk 5-0 sutures, in alternative</td>
		</tr>
		<tr>
			<td>Medbond Tissue Glue</td>
			<td>Stoelting</td>
			<td>50479</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>E8284</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Histolab</td>
			<td>01370</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (30G beveled needle)</td>
			<td>Hamilton</td>
			<td>80300</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>Aspen Nordic</td>
			<td>520455</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse head holder</td>
			<td>Narishige International</td>
			<td>SGM-4</td>
			<td>With mouth piece for inhalational anhestetics. Alternatively, use a stereotactic frame</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>15009-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Aesculap</td>
			<td>GT420</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile absorption spears</td>
			<td>Fine Science Tools</td>
			<td>18105-01</td>
			<td>Sterile cotton swabs are also a good option</td>
		</tr>
		<tr>
			<td>Surgical separator</td>
			<td>World Precision Instrument</td>
			<td>501897</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Viscotears</td>
			<td>Bausch&#38;Lomb Nordic AB</td>
			<td>541760</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of leptomeningeal cells modification via intracisternal injection

此处概述的协议描述了如何通过蓄水池安全、手动注入溶液，同时消除对底层帕伦奇马的损害风险。以前发布的协议建议使用直针，应降低至从杜面表面到最大 1-2 毫米。一旦硬膜被刺穿，阻力突然下降，使得很难将针头保持在稳定的位置。相反，我们的方法使用一根针弯曲在尖端，可以稳定在头骨的骨骼上，从而防止注射器在透过杜拉膜后渗透到组织中。该过程简单明了，可重复，不会在操作的动物中引起持久的不适。我们描述了血管脑蛋白酶细胞遗传命运图的背景下的宫内注射策略。此外，同样的技术也可以用来解决广泛的研究问题，例如通过基因消融这些现象，探讨利普托明病在神经发育和细菌性脑膜炎传播中的作用。此外，该程序可与自动输注系统相结合，实现持续输送，并通过注射荧光标记分子跟踪脑脊液运动。

在在Cx30促进剂13下表达CreER的转基因小鼠体内注射内毒素，以及一个可诱导的荧光报告器允许对瘦蛋白细胞进行特定重组，而无需在皮层中标记邻近的Cx30表达表面和帕伦奇质的天体细胞（图1）。为了获得进入蓄水池，麻醉动物的身体和头部以大约120°的角度定位，从而允许其颈部背部被拉伸（图1A）。</s...

Watch this Scientific Journal Video about Induction of Leptomeningeal Cells Modification Via Intracisternal Injection at JoVE.com

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

我们描述了一种宫内注射，在头尖弯曲的针头可以稳定在头骨上，从而消除了对底层腹腔损伤的风险。该方法可用于脑膜蛋白酶细胞的基因命运图谱和操作，以及跟踪脑脊液运动。

通过宫内注射诱导利普托门宁皮细胞修饰

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the ...

我们的外科手术使钩端细胞的特定基因编辑能够研究它们在许多生理和病理条件下的功能，如神经发育和细菌性脑膜炎的传播。为了尽量减少内西芬在内毒性分娩期间的损伤，我们使用弯曲的针头固定在头骨的骨边上，以防止其深入组织。该程序可用于通过功能实验的丧失和增益来探寻表达钩端细胞的基因的作用。它也可以被采用来跟踪脑脊液流。这种方法的视觉演示将确保成功识别注射部位的针刺注射部位，并了解如何将针头固定到腹骨上。首先准备一个汉密尔顿注射器与30量斜针注射。使用钳子将针头从尖端弯曲到 30 度三毫米。接下来，将内西芬稀释在10%DMSO中，浓度为每毫升1毫克，然后回填注射器。在异氟体室中麻醉动物。然后调整鼠标头架，使喉舌与手术台表面大约在 30 度角，将动物的头固定到支架上。为了改善对西斯特纳磁石的可访问性，将动物的身体定位在距离桌子表面约30度的位置，头部向下倾斜，这将与身体的其余部分建立120度的角度，并延伸颈部的后背，以方便接触西斯特纳磁体。一旦动物被正确定位，应用眼科软膏，剃光其颈部的背部，用酒精湿巾和贝塔丁消毒区域。使用手术剪刀，使中线切口从腹骨的水平开始，并延伸到后方。轻轻地分离表面的结缔组织和颈部肌肉，从中线侧身拉与细尖钳子，这将暴露的硬膜覆盖西斯特纳磁石。放置一个小的手术分离器，以便在整个手术过程中实现西斯特纳磁石的可视化。识别腹骨的骨质，并立即将先前弯曲的针头插入下方。一旦杜拉被打压，让针头弯曲的尖端穿透表面之下，轻轻将注射器向上拉，并平行于动物的身体，从而确保更好的稳定性。缓慢注射化合物，避免对脑脊液的自然流动造成干扰。注射后，让针头在内休息一分钟，然后用细尖钳小心地取出。用几滴氰丙烯酸酯粘合剂关闭皮肤，并在注射部位涂抹局部麻醉剂。将动物从支架上取下，放在加热垫上的清洁笼子里。然后确保监测动物，直到它恢复知觉。在Cx30启动子下表达CreER的转基因小鼠内皮注射内西芬，一种可教育的荧光报告器允许对钩端细胞进行特定的重组，而无需标记相邻的Cx30表达表面星细胞和皮层中皮层的面膜星细胞。此注射方案利用脑脊液的生理运动，使内毒素芬溶液分布在整个亚布拉奇体空间，以有效地重组覆盖嗅球、皮层和小脑的钩端细胞。该解决方案不会跨越脑脑膜屏障或与白细胞的天体利细胞接触，而不是通过口服治疗进行全身治疗。通过Pdgfr-alpha反应物识别后钩端膜细胞的重组，而表达Gfap的表面和腹膜星细胞仍未标记。尝试此实验时，必须找到注射部位进行针注。此视觉演示提供了执行该过程的技术知识，同时最大限度地降低了破坏其下神经组织的风险。基因消融研究可以用这种技术进行，以研究钩端细胞在皮质生成和脑脊液形成调节中的作用，并确定在亚巴氏体空间中传播细菌的其他位点。

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在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

用于访问视网膜下腔的另类和验证注射法<i&gt;通过</i&gt;一经巩膜后路

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

在Vivo直接重新编程居民胶质细胞到内神经通过病毒载体的脑内注射

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Brain Death Induction in Mice Using Intra-Arterial Blood Pressure Monitoring and Ventilation via Tracheostomy

使用动脉内血压监测和通气通过气管切除术的小鼠脑死亡诱导

While both living donation and donation after circulatory death provide alternative opportunities for organ transplantation, donation after donor brain ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

穿梭盒测定作为使用成人斑马鱼进行学习和记忆研究认知评估的关联学习工具

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

成人皮层的器官型培养作为人类干细胞移植和验证的 离体 模型

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...