这项工作得到了中国国家自然科学基金 (81650110527、81371400) 和中国国家重点基础研究项目 (2013CB530900) 的支持。

所有动物实验都是按照神经科学学会 (美国) 和复旦大学 (中国上海) 伦理委员会的政策进行的。这个程序是为非生存手术。
1. 脑脊液采集装置的设置
<ol><li>使用微拉拔玻璃毛细管 (内径为0.75 毫米, 外径1.0 毫米) (如刘et所示)。10;锐毛毛细管显示在图 1中)。</li>
	<li>将锐尖的玻璃毛细管放入牢固安装在机器人上的毛细管支架上 (图 1A)。</li>
	<li>在解剖显微镜下用直钳折断锐毛毛细管的尖端, 使断尖的内径约为10-20 µm (图 1C)。</li>
	<li>将细管和注射器连接到毛细管支架的另一端, 并用三通阀将薄壁管与注射器相连。</li>
	<li>将三路阀门打开, 然后将注射器从注射器中吹出空气, 通过油管向玻璃毛细管排放, 以排出任何污染物, 并在毛细管管内产生正压。</li>
	<li>重复这几次通过不连接和重新连接注射器到三路阀门, 并提请注射器100-200 µL 之前, 注射器的最后重新连接与三通阀 (图 1B)。</li>
	<li>将机器人与玻璃毛细管移动到侧面以防止在小鼠解剖过程中造成损伤。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56774/56774fig1.jpg"> 图1。显微镜, 机器人, 毛细管设置.(A) 显微镜和机器人与毛细管连接安装, 以及更近的图像 (B) 连接的注射器和三路阀门打开 (这里显示) 或关闭管道, 和 (C) 一个完整 (右) 和折断 (左)毛细管尖端准备脑脊液收集。一旦断裂, 毛细管的尖端应该有一个内径为0.75 毫米, 外径为1.0 毫米.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56774/56774fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 小鼠解剖
<ol><li>麻醉鼠标。 注: 对于本协议, C57BL/6 和淀粉样蛋白/早老素 1 (APP/PS1) 转基因 (阿尔茨海默病小鼠模型) 小鼠使用和麻醉4% 水合氯醛 (在dH2 0) 在10µL/克小鼠体重通过腹腔注射.</li>
	<li>当完全麻醉, 确保鼠标被充分麻醉。对脚趾捏的撤退反应, 稍微延长后肢和捏脚趾和观察撤退反应, 是为了确保足够的麻醉。用捏所有四只四肢检查;如果没有反应, 鼠标被适当麻醉。用解剖剪刀 (图 2A) 从眼睛上方切开和修剪鼠标头部后部的毛发, 从耳朵到颈部底部。或者, 剪刀或脱毛奶油可以用来帮助脱毛。</li>
	<li>使用鼠标适配器来保护鼠标头 (头向后朝上, 鼻子指向45°,图 2A)。在鼠标的耳朵和眼睛之间固定耳条并收紧。请确保头部无法移动, 并在适配器中通过轻轻向下向下推到鼠标头部而使其牢固地固定。</li>
	<li>在做手术切口前, 定期检查戒断反应, 并定时检查以保证手术面的麻醉。视觉上注意到在过程中呼吸速度的任何变化, 以表明麻醉的飞机的变化。一个干净的, 无菌的, 技术可以用于这种短期的非生存手术。使用剪刀和弯曲钳, 穿过皮肤和第一层肌肉, 直到头骨的底部暴露只有一层薄薄的肌肉。 注: 通过将老鼠的皮肤穿过颈部的背部, 然后将皮肤从头骨中间切开到老鼠的耳朵和眼睛之间。然后, 皮肤和组织可以被拉出, 并在麦格纳的范围外。</li>
	<li>在解剖显微镜下观察时, 用钳子仔细解剖头骨底部的最后一层肌肉, 并将这些肌肉层移动到侧面和远离感兴趣的区域。如果有出血, 使用棉花芽去除和帮助止血。</li>
	<li>一旦硬脑膜在大池被暴露 (三角形的形状与通常1-2 大血管在该地区运行; 无论是血管的一侧或之间的毛细血管插入和脑脊液收集,图 2B), 使用湿棉芽要擦拭膜清洁, 然后用干棉芽干燥该区域。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56774/56774fig2.jpg"> 图2。鼠标和解剖设置有代表性的图像毛细管插入脑脊液收集.设置用于脑脊液提取的鼠标 (A) 将剃掉的头夹在用于解剖的地方, (B) 详细的图像 (10x 视图) 的解剖小鼠硬脑膜覆盖池麦格纳 (虚线箭头显示一个血管贯穿该地区和实心箭头显示区域最佳的毛细管插入)。详细图像 (10x 视图) (C) 锐尖的玻璃毛细管对准麦格纳的硬脑膜, (D) 毛细管几乎刺穿硬脑膜, 与一些硬脑膜的阻力, 和 (E) 毛细管尖端挖掘通过硬脑膜收集脑脊液。脑脊液应该是一个清澈的液体收集在玻璃毛细管 (虚线箭头)。每个显微镜图像右下角的所有刻度条均显示为100µm.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56774/56774fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
3. 麻醉鼠脑脊液采集
<ol><li>将玻璃毛细管与鼠标头的背面对齐, 使锐化点刚好位于膜的后面30-45 °角 (图 2C)。</li>
	<li>注射一次或几次, 并提请注射器100-200 µL (以确保没有污染物, 并有负压在玻璃毛细管)。</li>
	<li>使用机器人, 将毛细管尖端移动到膜上, 直到能看到电阻, 但不能刺穿它 (图 2D)。</li>
	<li>一旦在毛细管和膜的尖端之间出现阻力, 通过敲机器人的控制, 轻轻地敲击毛细管通过膜。用显微镜观察毛细血管穿刺到麦格纳池的锐尖点。一旦穿刺孔被刺穿 (图 2E), 脑脊液应自动被抽出到毛细管管中。</li>
	<li>留下毛细血管慢慢收集脑脊液。如果脑脊液停止抽出, 抽出少量的注射器缓慢 (~ 50 µL), 以创造更多的负压毛细管, 以提取脑脊液。 注: 或者, 毛细血管可以轻轻地从大脑中缓慢拉出使用精细的机器人控制, 让脑脊液再次流入毛细血管。总金额为10-15 µL 的脑脊液 (3-4 厘米的毛细管) 采取〜10-30 分钟, 偶尔20µL 或更多的可以收集。虽然并非绝对必要, 但建议在脑脊液收集过程中使用热垫。脑脊液采集是在室温下 (摄氏23摄氏度) 进行的。</li>
	<li>一旦需要的脑脊液被收集, 关闭油管通过转移三路阀门关闭 (停止绘制脑脊液)。</li>
	<li>取出与油管相连的注射器, 然后打开并关闭油管 (以在毛细管、油管和毛细管外的区域产生均衡压力)。用三路阀门重新连接注射器, 但不要把注射器扎起来。</li>
	<li>使用机器人轻轻地从鼠标中取出玻璃毛细管。</li>
	<li>将收集管 (1.5 毫升管内与1µL 的20x 蛋白酶抑制剂) 置于玻璃毛细管的锐尖点下。</li>
	<li>打开油管, 轻轻地扎进注射器: 脑脊液应该流出毛细血管, 进入收集管。</li>
	<li>收集后, 快速离心机 (脉冲旋转为 5 s 以最大速度使用微型离心机) 脑脊液混合样品与蛋白酶抑制剂在底部的收集管。 注: 脑脊液样品可以 aliquoted, 然后储存以进一步分析-80 摄氏度。其余的老鼠可以解剖其他组织, 如大脑和脊髓。老鼠在这个阶段还活着, 如果需要, 也可以收集血液。</li>
</ol>

<ol>
	<li>Anoop, A., Singh, P. K., Jacob, R. S., Maji, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF+Biomarkers+for+Alzheimer's+Disease+Diagnosis.">CSF Biomarkers for Alzheimer's Disease Diagnosis.</a> Int. J. Alzheimers. Dis. 2010, 1-12 (2010).</li><li>Blennow, K., Hampel, H., Weiner, M., Zetterberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+and+plasma+biomarkers+in+Alzheimer+disease.">Cerebrospinal fluid and plasma biomarkers in Alzheimer disease.</a> Nat. Rev. Neurol. 6 (3), 131-144 (2010).</li><li>Schoonenboom, N. S. 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=Cerebrospinal+fluid+markers+for+differential+dementia+diagnosis+in+a+large+memory+clinic+cohort.">Cerebrospinal fluid markers for differential dementia diagnosis in a large memory clinic cohort.</a> Neurology. 78 (1), 47-54 (2012).</li><li>Molinuevo, J. 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=The+clinical+use+of+cerebrospinal+fluid+biomarker+testing+for+Alzheimer's+disease+diagnosis:+A+consensus+paper+from+the+Alzheimer's+Biomarkers+Standardization+Initiative.">The clinical use of cerebrospinal fluid biomarker testing for Alzheimer's disease diagnosis: A consensus paper from the Alzheimer's Biomarkers Standardization Initiative.</a> Alzheimer's Dement. 10 (6), 808-817 (2014).</li><li>Fagan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF+biomarkers+of+Alzheimer's+disease:+impact+on+disease+concept,+diagnosis,+and+clinical+trial+design.">CSF biomarkers of Alzheimer's disease: impact on disease concept, diagnosis, and clinical trial design.</a> Adv. Geriatr. 2014, 1-14 (2014).</li><li>Ramautar, 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=Metabolic+profiling+of+mouse+cerebrospinal+fluid+by+sheathless+CE-MS.">Metabolic profiling of mouse cerebrospinal fluid by sheathless CE-MS.</a> Anal. Bioanal. Chem. 404 (10), 2895-2900 (2012).</li><li>Liu, L., Herukka, S., Minkeviciene, R., Vangreon, T., Tanila, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+observation+on+CSF+A&#946;42+levels+in+young+to+middle-aged+amyloid+precursor+protein/presenilin-1+doubly+transgenic+mice.">Longitudinal observation on CSF A&#946;42 levels in young to middle-aged amyloid precursor protein/presenilin-1 doubly transgenic mice.</a> Neurobiol. Dis. 17 (3), 516-523 (2004).</li><li>Schelle, 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=Prevention+of+tau+increase+in+cerebrospinal+fluid+of+APP+transgenic+mice+suggests+downstream+effect+of+BACE1+inhibition.">Prevention of tau increase in cerebrospinal fluid of APP transgenic mice suggests downstream effect of BACE1 inhibition.</a> Alzheimer's Dement. , (2016).</li><li>You, J. -. S., Gelfanova, V., Knierman, M. D., Witzmann, F. A., Wang, M., Hale, 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=The+impact+of+blood+contamination+on+the+proteome+of+cerebrospinal+fluid.">The impact of blood contamination on the proteome of cerebrospinal fluid.</a> Proteomics. 5 (1), 290-296 (2005).</li><li>Liu, L., Duff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Technique+for+Serial+Collection+of+Cerebrospinal+Fluid+from+the+Cisterna+Magna+in+Mouse.">A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse.</a> J. Vis. Exp. (21), (2008).</li><li>Maia, L. 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=Changes+in+amyloid-&#946;+and+Tau+in+the+cerebrospinal+fluid+of+transgenic+mice+overexpressing+amyloid+precursor+protein.">Changes in amyloid-&#946; and Tau in the cerebrospinal fluid of transgenic mice overexpressing amyloid precursor protein.</a> Sci. Transl. Med. 5 (194), 194re2 (2013).</li><li>Oshio, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+cerebrospinal+fluid+production+and+intracranial+pressure+in+mice+lacking+choroid+plexus+water+channel+Aquaporin-1.">Reduced cerebrospinal fluid production and intracranial pressure in mice lacking choroid plexus water channel Aquaporin-1.</a> FASEB J. , (2004).</li></ol>

作者没有什么可透露的。

本协议详细描述了一种技术, 它改进了现有方法10脑脊液收集, 以最大限度地减少血液污染, 并允许大量收集脑脊液 (平均约10-15 µL 可获得) 从小鼠。当打破毛细管尖端, 毛细管的尖端不应该太小 (因为然后脑脊液将被提取非常缓慢) 或太大 (将不够罚款, 以收集脑脊液和组织可以成为在毛细管)。在解剖脑脊液收集区域时应注意: 应停止任何出血, 并将玻璃毛细管的插入区域与 dH2</s...

脑脊液是一种有价值的体液, 用于分析神经科学研究。脑脊液主要由血浆组成, 含有少量细胞 (没有红细胞, 只有少数白细胞), 几乎无蛋白质。它是与中枢神经系统 (CNS) 紧密接触的液体之一, 它可以将许多电解质从大脑和脊髓传给外围系统。在人类中, 可以收集脑脊液样本以帮助诊断疾病或临床试验中的研究目的, 因为脊椎穿刺 (或腰椎穿孔) 是一种轻微的侵入性手术: 脑脊液可以反映中枢神经系统的变化, 而无需直接访问这些组织.因此, 近年来, 为临床研究目的, 脑脊液样本已获得从神经退行性疾病的患者, 如阿尔茨海默病和其他痴呆1,2,3。有许多生物标志物的研究已经开发使用脑脊液样本, 以潜在的帮助诊断疾病的诊所2,3。然而, 对于这些化验的可靠性, 有很多争论, 以产生一致的, 敏感的结果, 专门诊断疾病4,5。因此, 有必要发展更好的检测和指标, 可以在脑脊液中找到, 以帮助生产一种标准技术, 以更大的灵敏度和特异性诊断神经变性疾病。由于人体脑脊液样本在疾病中的潜在重要性, 在神经科学研究中采集啮齿目动物的脑脊液也很有兴趣。
小鼠是生物学和医学研究中的重要动物, 在人类临床试验之前, 允许对潜在的治疗化合物和概念证明研究进行测试。然而, 在小鼠很难获得脑脊液标本由于它接近大脑在一个小动物, 因为通常的脑脊液收集方法是通过大池获得, 在小脑和延髓背表面之间的开放。这导致采集脑脊液标本困难, 因为这一地区很难解剖, 并与血管接近, 增加了血细胞污染的风险。由于这些困难, 大多数研究人员只能获得少量的脑脊液进行分析 (通常说是5-7 µL), 并且血液细胞对脑脊液样品的污染是分析6、7、8的主要关注因素。,9. 血液污染会使结果模糊不清, 并不能真正反映中枢神经系统的状态。此外, 有限样本收集可以影响研究, 因为通常的数量从小鼠是足够的只有一个测量 (重复或三个) 使用酶联免疫吸附试验 (ELISA)。因此, 脑脊液标本通常是从多个小鼠汇集, 以便有足够的样本来运行多种化验。为大量、无污染的小鼠脑脊液收集提供了一个协议, 这对改善利用啮齿动物进行神经科学研究是有益的。
在本议定书中, 详细描述了从麻醉小鼠中大量 (平均10-15 µL) 收集脑脊液的技术, 并改进了目前已知的脑脊液收集方法, 以最大限度地减少血液中的污染10。一个强健的脑脊液收集协议将有助于开发基于脑脊液的生物标志物化验, 这可以用来帮助诊断疾病, 并改进对影响中枢神经系统疾病的机制的研究。

<table><tbody><tr><td>Chloral hydrate (used as anesthetic)</td><td>Sinopharm Chemicals Reagen Co. Ltd.</td><td>30037517</td><td>CAS number 302-17-0.</td></tr><tr><td>Dissecting scissors</td><td>66 vision technology</td><td>54002</td><td></td></tr><tr><td>Dissecting curved forceps</td><td>66 vision technology</td><td>53072</td><td></td></tr><tr><td>Dissecting straight forceps</td><td>66 vision technology</td><td>53070</td><td></td></tr><tr><td>Mouse adapter (with ear bars)</td><td>Made in-house.</td><td>N/A</td><td>Similar equipment available from World Precision Instruments.</td></tr><tr><td>Dissecting microscope</td><td>Meiji Labax</td><td>Model 15381</td><td></td></tr><tr><td>Micromanipulator</td><td>World Precision Instruments</td><td>M3301</td><td></td></tr><tr><td>Magnetic base for micromanipulator</td><td>Kanetec</td><td>MB-K</td><td></td></tr><tr><td>Glass capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>Model P-1000</td><td></td></tr><tr><td>Syringes (1ml)</td><td>Tansoole</td><td>02024692</td><td>For 1ml.</td></tr><tr><td>Microtubes (1.5ml)</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>Protease inhibitor Cocktail Set III EDTA-free</td><td>Calbiochem</td><td>539134</td><td></td></tr><tr><td>Human A&amp;beta;42 ELISA kit</td><td>Invitrogen</td><td>KHB3441</td><td></td></tr><tr><td>Piping (teflon tubing)</td><td>World Precision Instruments</td><td>MMP-KIT</td><td>Obtained from a microinjection kit and attached to the capillary holder and syringe.</td></tr><tr><td>Mini centrifuge</td><td>Tiangen Biotech</td><td>OSE-MC8</td><td></td></tr><tr><td>Cotton buds</td><td>Obtained from any household store/pharmacy.</td><td>N/A</td><td></td></tr></tbody></table>

an improved method for collection of cerebrospinal fluid from anesthetized mice

脑脊液 (CSF) 是一种有价值的体液, 用于分析神经科学研究。它是最接近中枢神经系统的液体之一, 因此可以用来分析大脑或脊髓的患病状态而不直接进入这些组织。然而, 小鼠由于与血管的亲近而难以从大池获得, 这通常会污染样本。小鼠的脑脊液采集面积也很难解剖, 而且通常只有小样本 (最多5-7 µL 或更少)。该协议详细描述了一种技术, 改进现有的收集方法, 以尽量减少血液污染, 并允许大量收集脑脊液 (平均10-15 µL 可以收集)。这种技术可以与其他解剖方法, 从小鼠组织收集, 因为它不会影响任何组织在脑脊液提取。因此, 大脑和脊髓不受这种技术的影响, 并保持不变。随着脑脊液样品的收集和纯度的增加, 更多的分析可以用来进一步帮助神经科学研究, 更好地了解影响大脑和脊髓的疾病。

使用此处概述的过程 (图 1和图 2), 立即在毛细管中收集的 CSF 应该是清晰的 (图 2E), 而不是粉红色或红色。如果在毛细管中收集的液体中有粉红色到红色的色调, 那么就会有血液污染。
以该方法采集的脑脊液样品的应用为例, 用 ELISA 法测定了蛋白质淀粉样蛋白β...

Watch this Scientific Journal Video about An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice at JoVE.com

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

本协议描述了一种改进的技术, 用于丰富的脑脊液 (CSF), 不污染血液。随着样品的收集和纯度的增加, 可以通过脑脊液进行更多的分析, 进一步了解影响大脑和脊髓的疾病。

一种改进的麻醉小鼠脑脊液采集方法

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

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Research

JoVE Journal

Neuroscience

61.4K 次观看.  University of Chinese Academy of Sciences, Chinese Academy of Science. 本协议描述了一种改进的技术, 用于丰富的脑脊液 (CSF), 不污染血液。随着样品的收集和纯度的增加, 可以通过脑脊液进行更多的分析, 进一步了解影响大脑和脊髓的疾病。

视频: An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

一个串行收集脑脊液从池鼠标麦格纳技术

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

科研

生物学

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

脑脊液分离解剖大脑皮质外植体使用的啮齿动物胚胎

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

神经科学

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

将套管植入啮齿动物的麦格纳池中

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

小鼠体内合成蛋白的定量测量

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous ...

绘制脑血流和电场图:小鼠的电极放置

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

作者聚焦:基于刺激的方法改善阿尔茨海默病模型中的脑血流 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...

同时记录脑电图的大鼠脑脊液和血液

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

作者聚焦:获取高质量的 CSF 和血液样本以发现癫痫生物标志物 

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

在脑电图记录期间从大鼠的侧尾静脉采集脑脊液和血液

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...

一种改进的麻醉小鼠脑脊液采集方法

摘要

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此视频中的章节

一个串行收集脑脊液从池鼠标麦格纳技术

脑脊液分离解剖大脑皮质外植体使用的啮齿动物胚胎

将套管植入啮齿动物的麦格纳池中

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

小鼠体内合成蛋白的定量测量

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

在脑电图记录期间从大鼠的侧尾静脉采集脑脊液和血液