这项工作得到了诺德基金会和国立神经疾病和中风研究所 (M.N.) 的支持。a.l.r. x 和 S. R 是博士后奖学金的接受者和 Lundbeck 基金会的博士学位。

所有程序均按照欧洲 2010/63/欧盟关于动物研究的指令进行, 并经丹麦环境和食品部 (2015-15-0201-00535) 的动物实验委员会批准。
1. 插管程序
<ol><li>套管准备 注意: 避免用无菌手套接触套管。<ol>
<li>用针架切断30G 牙针的斜面金属尖端。</li>
		<li>使用针架, 将斜面金属尖端 (大约0.3 厘米) 插入30厘米长的 PE10 油管 (聚乙烯油管 0.024 "OD x 0.011" ID), 填充 aCSF (126 毫米氯化钠, 2.5 毫米氯化钾, 1.25 毫米 NaH2PO4, 2 毫米 MgSO4, 2 毫米 CaCl2, 10 毫米葡萄糖, 26 毫米 NaHCO3;pH 值为 7.4, 当使用 95% O2和 5% CO2进行毒气时。</li>
		<li>使用装有30G 针的1毫升注射器 (30 克 x ½ "0.3 x 12") 冲洗套管与 aCSF。</li>
	</ol>
</li>
	<li>
手术 注意: 慢性厘米插管允许动物从手术过程中恢复。CM 注射是在套管植入后的第二天进行的, 重要的是可以在麻醉或醒着的动物中进行。由于这是一个康复手术, 程序应在无菌条件下进行。<ol>
<li>称鼠标 (C57BL/6JRj, 两性, 8 周), 并麻醉氯胺酮和甲苯噻嗪 (100 毫克/千克; 10 毫克/千克) 的混合物, 通过腹腔 (ip) 注射。如有必要, 在手术过程中建立小鼠半剂量氯胺酮 (50 毫克/千克)。或者, 对于慢性插管, 麻醉小鼠, 将其置于异氟醚诱导腔中 2.5-3% 异氟醚, 在大约1升/分 O2。在这种情况下, 使用鼻锥维持异氟醚麻醉在 1.5-2% 整个手术。</li>
		<li>当脚趾捏的反应停止, 呼吸变得缓慢和稳定, 把动物放在一个立体定向的框架上的加热垫。</li>
		<li>应用眼科软膏。必要时重复手术。</li>
		<li>剃掉老鼠的头部和颈部, 去掉毛皮, 用酒精拭子先消毒暴露的皮肤, 然后两次用洗必泰 (0.5%) 或碘溶液 (2%)。重复灭菌两次。 注意: 在剃须后, 改变手术悬垂以清除杂物和毛发。然后将动物披上, 保护不育的田地。</li>
		<li>对于慢性插管, 在切口部位分别管理 0.5-1 毫升利多卡因 (1 毫克/毫升和0.25 毫克/毫升) 皮下 (南卡罗来纳州)。管理丁丙诺啡 (0.05 毫克/千克) 用于术后镇痛。</li>
		<li>将鼠标固定在立体定向的框架中。在确保固定后, 无论是 intraural 或颧骨拱, 稍微倾斜头部, 使其形成一个角度的120°的身体 (图 1E)。</li>
		<li>发现头骨的一部分, 立即突出的颈部肌肉-枕嵴。用一双镊子抬起上面的皮肤, 在中线处切开大约1厘米的杏仁状的皮肤。使用棉签或眼矛控制任何结果出血。</li>
		<li>以枕嵴为参考点, 将浅层结缔组织拉出, 露出颈部肌肉。</li>
		<li>小心地将钳位放在中间的切口部位, 在前-后轴处, 将其分开。每只手上都有一对弯曲的镊子, 在头骨底部的中间加入尖端, 把肌肉拉到一边。 注意: 这应该暴露 CM, 它出现作为一个微小的倒置的三角, 概述由小脑上面和髓质下面, 在半透明的硬脑膜膜 (图 1B 和 1C) 之后。</li>
		<li>使用手术眼矛或棉签, 擦拭覆盖 CM 的硬脑膜膜。</li>
	</ol>
</li>
	<li>
插入套管入 CM
	<ol>
<li>从 aCSF 的注射器中取出套管, 使30G 针附着在油管的后端。</li>
		<li>将30G 针连接到注射器泵上的蒸馏水填充100µL 注射器上。</li>
		<li>用注射器泵取出空气, 在套管中插入大约1厘米的气泡。</li>
		<li>使用注射器泵, 提取 12 ul 的预期脑脊液示踪剂进入套管。</li>
		<li>抓住套管, 用脑脊液示踪剂, 靠近管盖针, 用一双弯曲的镊子握在支配手上。将非显性手的中指放在对面的耳条上, 并保持其稳定, 供以后用作套管的休息用。</li>
		<li>将套管插入与鼠标头相对的45°角, 通过硬脑膜所看到的三角形的角度, 将其传递到 CM 的中心。避免小脑或髓质的任何穿透。确保针只插入到深度为 1-2 毫米,即到斜角完全位于硬脑膜下的点。松开镊子持有套管, 并让套管休息的非显性的手。 注: 牙针的斜面端将需要使用一些力量刺穿硬脑膜膜覆盖 CM。</li>
		<li>如有必要, 使用手术眼矛或棉签拭去任何脑脊液渗漏。</li>
		<li>将 2-3 滴氰胶水放在套管周围的硬脑膜膜上。添加一滴胶水加速器, 立即固化胶水。用牙科水泥 (大约0.5 毫克) 和氰胶 (3-5 滴) 的混合物盖住头骨和针头。紧接着, 应用一滴胶水加速器来治疗。</li>
		<li>对于慢性插管, 切油管 (留约 2-3 厘米连接到套管), 并密封它与外科焊接保持颅内压 (ICP) 水平, 通过防止脑脊液泄漏通过油管。</li>
		<li>用于慢性插管, 管理卡洛芬 (5 毫克/千克;</li>
		<li>对于慢性插管, 把鼠标放在笼子里, 把它放在加热垫上保持体温, 直到动物完全从麻醉中恢复。 注意: 动物应该单独存放在笼子里, 以确保套管完好无损。通过将鼠标放在纸巾或其他固体基板上, 确保鼻子清晰的床上用品。 注: 第二天, 当动物从手术过程中恢复为套管插入, 他们可以注射脑脊液示踪剂。在麻醉下注射, 管理氯胺酮/甲苯噻嗪的混合物 (100 毫克/千克; 10 毫克/千克, 分别; ip), 然后继续执行2 节中介绍的步骤。对于唤醒动物的注射, 请继续执行3节。</li>
	</ol>
</li>
</ol>2. 麻醉动物急性植入 CM 导管注射脑脊液示踪剂
注: 在麻醉动物的急性植入性 CM 套管注射脑脊液示踪剂后, 在上一节步骤8后立即进行脑脊液示踪剂注射, 如下所述。
<ol><li>使用注射器泵, 以 1 ul/分5或10分钟的速度开始注射到脑脊液示踪剂的 CM 中, 从而分别产生5µL 或10µL 的总体积。在注射结束时, 允许脑脊液追踪器在整个大脑中循环30分钟, 不受干扰的套管。</li>
	<li>30分钟后, 切断连接到套管的油管 (大约4厘米的距离从针尖) 和密封它的末端使用外科焊接。</li>
	<li>在深度麻醉下, 用斩首弄死动物。快速解剖大脑和修复组织通过浸泡在4% 多聚甲醛 (粉煤灰) 稀释磷酸盐缓冲盐水 (PBS; 0.01M; pH 值 7.4) 隔夜 (o/n) 在4摄氏度。</li>
</ol>3. 通过长期植入的 CM 套管在醒动物体内注射脑脊液示踪剂
<ol><li>使用注射器泵, 提取7µL 或12µL 所需的脑脊液示踪剂组成的套管约30厘米的 PE10 导管与斜角牙科针尖0.5 厘米。</li>
	<li>轻轻地抑制动物和切割约1厘米的油管附加到套管。</li>
	<li>仍然在温和的限制下, 快速连接的套管填充脑脊液示踪剂到 CM 植入套管。</li>
	<li>使用注射器泵, 开始注射到脑脊液示踪剂的 CM 的速度为1µL/分钟. 注射7µL 或12µL, 以达到最终容积的脑脊液注射示踪5µL 或10µL, 从而弥补 aCSF 留在植入套管。在注射结束时, 允许脑脊液追踪器在整个大脑中循环30分钟, 不受干扰的套管。确保油管在注射和循环期间保持连接。</li>
	<li>脑脊液示踪循环结束时, 通过斩首进行安乐死, 保证动物被深深麻醉。快速解剖大脑和修复组织通过浸泡在4% 多聚甲醛 (粉煤灰) 稀释磷酸盐缓冲盐水 (PBS; 0.01M; pH 值 7.4) 隔夜 (o/n) 在4摄氏度。</li>
</ol>

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作者没有什么可透露的。

我们已经提出了一个协议, 描述了一个详细的程序, 一池的麦格纳插管 (CMc) 提供了一个简单的方法, 提供标签的分子到脑脊液室。CMc 允许随后的脑脊液动力学可视化, 无论是在体内和前体,使用不同的成像方式或组织学。
CMc 技术的主要优点之一在于它可以直接进入蛛网膜下腔, 而无需通过开颅手术暴露大脑。通过不需要颅骨窗口或大脑实质的穿透针尖, CMc 允许分子进入脑脊液室和评估 glymphatic 系统的微创程序, 只有短暂的干扰颅内压 (ICP)。
值得注意的是, 注射到 CM 是脑脊液的主要来源, 脉络膜树脂位于心室系统 (侧, 第三和第四脑室) 的下游。从侧脑室, 脑脊液流向第三脑室通过脑室孔 (蒙罗) 和从第三至第四脑室通过脑渡槽 (Sylvius 的渡槽) 到脑干和脊髓 (审查在3).脑脊液通过中位孔径 (或 Magendie 孔) 通过 CM 到达蛛网膜下腔, 因此 CMc 注射液绕过整个心室系统。然而, 虽然这可能是一个问题, 在一些模型的脑脊液/艾滋病的动态通过心室, 直接注射示踪剂进入心室需要侵入性手术程序, 如钻孔的头骨窗口, 并应用心室注射极大地扰乱了 ICP13。同样, 将示踪剂注入到蛛网膜下腔内的13、14中, 我们的手就能在 paravascular 空间中废除脑脊液示踪剂的通量。相比之下, 即使 CMc 需要穿刺硬脑膜膜, ICP 只是短暂的扰动, 并迅速恢复2。
使用 CMc, glymphatic 活动可以测量在麻醉动物急性 CMc, 以及在清醒的动物, 观察24小时的恢复期后, 套管植入。急性 CMc 适合与2光子成像相结合, 它提供有关皮层内 glymphatic 活动的详细信息, 深度约200µm1,2。重要的是, 急性 CMc 也提供支持无偏 MRI 研究的优势, 在那里追踪器分布动态地跟踪, 相对于在启动脑脊液示踪剂注射前获得的单个基线图像15,16,17. 对于 MRI, 用于 CMc 的牙科针应用硼硅酸盐毛细管 (大约1厘米长, 大约20µm 的尖端直径) 代替 PE 油管。
与急性插管相比, 慢性 CMc 允许实验者在自然睡眠或麻醉下, 以及在清醒的、自由移动的动物中, 对动物进行脑脊液示踪剂注射。这是一个关键因素, 因为 glymphatic 活动是高度依赖于国家的;在麻醉或睡眠中注射的动物中, 示踪物流入到实质中要大得多, 而不是在清醒状态1中注射的动物。此外, 长期植入套管的动物可以在其笼中接收脑脊液示踪剂, 从而尽量减少因压力和唤起对 glymphatic 活动的影响而造成的混杂因素。对于麻醉下的慢性注射, 建议采用氯胺酮/甲苯噻嗪 (分别为100毫克/千克; 10 毫克/千克) 的混合物。异氟醚在1.5% 以上的浓度诱发脑肿胀, 并没有加强 glymphatic 活动相比, 清醒状态1。注意, 在 cmc 植入后, 动物应单独安置, 以确保 CMc 植入的动物不会损害彼此的套管。此外, 由于 CMc 慢性植入术是一种恢复性手术, 应在无菌条件下进行, 动物应接受操作后镇痛药。
重要的是, CMc 可以作为一种方法, 提供脑脊液示踪剂在小鼠和大鼠, 对协议的最小修改。由于两种不同的心室和蛛网膜下腔的大小有差异, 应进行适当的麻醉药剂量, 并在大鼠中注射的脑脊液示踪剂的最大体积为30µL。
尽管它的程序简单, 一些训练和实践是需要的实验者成功地执行 CMc。由于 CM 在物种和个体动物之间的大小不同, 因此最好实践对其结构的识别。使用埃文斯蓝色 (2% 在 aCSF) 实践过程允许实验者确认正确的针插入。有时, 一艘船将直接位于 CM 中线, 因此应将针插入相邻的容器, 但尽量靠近中线。应注意这些病例, 以便以后确认示踪剂均匀分布, 尽管针尖的中心位置不居中。重要的是, 寰枕膜覆盖 CM 是机械强硬, 并应充分的压力, 以插入斜面针尖端。然而, 重要的是, 施加的压力不会导致针尖进入髓质或小脑。为了促进针插入到 CM, 动物的头应该倾斜向下在120°的角度相对于身体, 舒展膜。重要的是, 应注意不要因为头部屈曲而阻碍呼吸。如果针尖应进入小脑, 示踪剂将保留在组织中, 不能在整个蛛网膜下腔分布。髓质损伤通常是致命的, 而慢性插管的小脑损伤可能导致虚脱和动物行为的一般异常。为了尽量减少这种可能性的风险, 可以使用较小的斜角长度的针头。
当移动的颈部区域的肌肉, 覆盖硬脑膜膜插入套管到 CM, 出血可能发生。棉签可以用来吸收出血, 但也可以采用氯化铁溶液。氯化铁具有止血效果18, 并触发了颈部肌肉的加强周围的切口部位, 从而帮助获得正确插入的针到 CM. 氯化铁也干燥头骨和硬脑膜膜,为套管的附着力提供更好的表面。对于 CMc, 应用 1-2 滴氯化氯化铁溶液 (10%)(约1毫升) 成棉签, 并将颈部肌肉和枕嵴。然而, 局部氯化铁可能渗透到脑脊液中, 对大脑稳态的影响未知。如果使用氯化铁是一个值得关注的问题, 你可以使用伤口拉钩保持打开切口部位。使用氰胶后, 小心清除伤口拉钩, 避免意外附着在切口部位。
CMc 是一种简单而又可重复的程序, 直接将分子送进脑脊液室。由于 CMc 是微创的, 它是 glymphatic 系统可视化的首选方法, 可以结合不同的成像方式, 如荧光和2光子显微镜或 MRI。因此, CMc 是研究流体动力学的一个很好的工具, 即脑脊液和, 以及脑液间隙。由于 glymphatic 系统的宏观覆盖, CMc 有可能被用来提供大脑范围内的分子。

脑脊液 (CSF) 将中枢神经系统 (CNS) 沐浴在整个心室系统和蛛网膜下腔内, 这是一个解剖上定义的空间在连续体与脑室, 围绕大脑和脊髓。脑脊液的主要功能之一是提供从脑实质中清除代谢物和溶质的途径。通过最近发现的 glymphatic 系统1, 将大脑模拟到外围淋巴系统, 方便了清除。在这里, 我们描述和讨论的是一个微创的方法, 以直接提供分子进入脑脊液池的麦格纳插管 (CMc)。CMc 是研究 glymphatic 功能的关键方法。此外, CMc 也可以应用于脑脊液动力学研究和快速, 大脑范围内的非血脑屏障 (BBB) 渗透分子进入脑实质, 沿周围空间。
CMc 通过中枢神经系统利用脑脊液运动动力学的生理原理, 将标记的示踪分子或药物传送到麦格纳池 (CM) 的脑脊液填充空间。分子通过插入到寰枕硬脑膜膜内的套管注入, 覆盖厘米. 然后通过 paravascular 空间1, 将脑脊液散装流进入脑实质。通过 CMc 注射的示踪剂或造影药剂跟随脑脊液的运动, 它允许评估脑脊液运动和 glymphatic 的流入, 量化的强度水平的标记分子进入大脑实质。CMc 兼容不同的成像技术, 包括荧光, 2 光子显微术, 磁共振成像 (MRI)。此外, 可以在体内或前体中执行此评估。重要的是, CMc 允许 glymphatic 系统的可视化在麻醉下或在自然睡眠期间, 并且在醒的, 自由地移动的动物。
CMc 技术可用于研究脑脊液中流体动力学的不同方面, 但已证明对研究 glymphatic 系统特别有用。Glymphatic 活动通过 aquaporin-4 (AQP-4) 水通道在胶质血管包裹 endfeet 的膜中拴在 periarterial 空间中, 推动脑脊液的对流流动。对流流使脑脊液和间质流体在脑实质中相互交换。通过 perivenous 空间2,3, 将含有代谢废物和溶质的 CSF 从脑实质中移除。最终, CSF 通过最近描述的硬脑膜淋巴管到达外围,4,5。对于清除有害废物代谢物 (如淀粉样蛋白β2), glymphatic 系统已被证明是至关重要的。进一步, glymphatic 清除在老化6, 在创伤性脑损伤以后7, 并且在糖尿病的动物模型8和阿尔茨海默病9。值得注意的是, glymphatic 活动是状态依赖性的, 在睡眠或麻醉中显示的活动显著高于清醒的1。事实上, 年轻的麻醉动物表现出最高的 glymphatic 活动。因此, 在研究其在健康和疾病中的作用时, glymphatic 活动的实验量化是至关重要的。
几项研究已经讨论了脑脊液动力学及其与间质流体的交换在脑实质。然而, 标记的分子被提供的方法是相当侵入性的, 触发脑实质损伤和颅内压的变化 (ICP) (见回顾10)。一些例子是脑室内或脑实质注射, 涉及开颅手术或钻孔在头骨的一个毛刺孔。这些过程已被证明可以改变 ICP, 从而中断 glymphatic 函数2。此外, 这种侵入性方法诱发 astrogliosis 和增加 AQP-4 免疫反应在脑实质受损地区及其周围的11,12。由于星形胶质细胞和 AQP-4 是 glymphatic 系统的关键元素, CMc 是其研究的选择方法。CMc 与更多侵入性程序相比, 其主要优点是保持完整的颅骨和脑实质, 避免 ICP 改变和 astrogliosis。因此, CMc 与不同的成像工具一起打开了广泛的可能性, 不仅研究 glymphatic 系统, 而且还有流动的动力学和机制的动态平衡, 以及动物模型的神经系统疾病。
该池 (CMc) 程序允许容易和直接进入脑脊液 (CSF)。通过注射不同的分子 (例如荧光示踪剂, MRI 造影药), 实验者可以跟踪他们的运动在脑脊液室和评估 glymphatic 系统的活动。下面的协议描述了急性 CMc, 在手术后立即注射, 和慢性套管植入, 其中动物从手术过程中恢复为以后注射。急性和慢性植入的最重要的区别是慢性植入允许研究醒鼠的 glymphatic 活动。

<table><tbody><tr><td>SOPIRA Carpule 30G 0.3 x 12mm</td><td>Kulzer</td><td>AA001</td><td></td></tr><tr><td>Polyethylene Tubing 0.024&amp;rdquo; OD x 0.011&amp;rdquo; ID</td><td>Scandidact</td><td>PE10-CL-500</td><td></td></tr><tr><td>30G x &amp;frac12;&amp;rdquo; 0.3 x 12 mm Luer-Lock</td><td>Chirana T. Injecta</td><td>CHINS01</td><td></td></tr><tr><td>Chlorhexidine 0.5% (chlorhexidine digluconate)</td><td>Meda AS</td><td>no catalogue number, see link in comments</td><td>http://www.meda.dk/behandlingsomrader/desinfektion/desinfektion-af-hud/klorhexidin-sprit-medic-05/</td></tr><tr><td>Alcohol Swab 70% Isopropyl Alcohol 30 x 60mm</td><td>Vitrex Medical A/S</td><td>520213</td><td></td></tr><tr><td>Viskoese Oejendraeber Ophtha</td><td>Ophtha</td><td>145250</td><td></td></tr><tr><td>Wooden applicator, Double cotton bud (&amp;Oslash; appr. 4 - 5 mm, length appr. 12 mm)</td><td>Heinz Herenz</td><td>1032018</td><td></td></tr><tr><td>Eye spears</td><td>Medicom</td><td>A18005</td><td></td></tr><tr><td>Ferric chloride 10% solution</td><td>Algeos</td><td>NV0382</td><td></td></tr><tr><td>Kimtech Science Precision Wipes Tissue Wipers</td><td>Kimberly Clark Professional</td><td>05511</td><td></td></tr><tr><td>Loctite Super Glue Precision 5g</td><td>Loctite</td><td>no catalogue number, see link in comments</td><td>http://www.loctite-consumer.dk/da/produkter/superglue-liquid.html&amp;nbsp;</td></tr><tr><td>Insta-Set CA Accelerator</td><td>Bob Smith Industries</td><td>BSI-152</td><td></td></tr><tr><td>Dental Cement Powder</td><td>A-M Systems</td><td>525000</td><td></td></tr><tr><td>Surgical weld&amp;nbsp;</td><td>Kent Scientific Corporation</td><td>INS750391</td><td></td></tr><tr><td>Hamilton syringe GASTIGHT&amp;reg; , 1700 series, 1710TLL, volume 100 &amp;mu;L, PTFE Luer lock</td><td>Hamilton syringes</td><td>1710TLL</td><td></td></tr><tr><td>LEGATO 130 Syringe pump</td><td>KD Scientific</td><td>788130</td><td></td></tr><tr><td>Paraformaldehyde powder, 95%</td><td>Sigma Aldrich</td><td>158127</td><td></td></tr><tr><td>Phosphate buffered saline (PBS; 0.01M; pH 7.4)</td><td>Sigma Aldrich</td><td>P3813</td><td></td></tr><tr><td>Ovalbumin, Alexa Fluor 647 Conjugate</td><td>Thermo Fisher Scientific</td><td>O34784</td><td></td></tr><tr><td>&lt;br/&gt; DAPI (diamidino-2-phenylindole) Solution (1 mg/mL)</td><td>Thermo Fisher Scientific</td><td>62248</td><td></td></tr><tr><td>Dextran, Fluorescein, 3000 MW, Anionic</td><td>Thermo Fisher Scientific</td><td>D3305</td><td></td></tr><tr><td>E-Z Anesthesia EZ-7000 Classic System</td><td>E-Z Systems</td><td>EZ-7000</td><td></td></tr><tr><td>Attane Isofluran 1000 mg/g</td><td>ScanVet</td><td>55226</td><td></td></tr><tr><td>Euthanimal 200mg/mL (sodium pentobarbital)</td><td>ScanVet</td><td>545349</td><td></td></tr><tr><td>Ketaminol Vet 100 mg/mL (ketamine)</td><td>Intervet International BV</td><td>511519</td><td></td></tr><tr><td>Rompin Vet 20 mg/mL (xylazin)</td><td>KVP Pharma + Veterin&amp;auml;r Produkte GmbH</td><td>148999</td><td></td></tr><tr><td>Xylocain 20 mg/mL (lidocain)</td><td>AstraZeneca</td><td>158543</td><td></td></tr><tr><td>Marcain 2.5 mg/mL (bupivacain)</td><td>AstraZeneca</td><td>123918</td><td></td></tr><tr><td>Bupaq Vet 0.3 mg/mL (buprenorphine)</td><td>Richter Pharma AG</td><td>185159&amp;nbsp;</td><td></td></tr></tbody></table>

cannula implantation into the cisterna magna of rodents

大池插管 (CMc) 是一个简单的程序, 使直接进入脑脊液 (CSF) 没有手术损伤的头骨或脑实质。在麻醉啮齿目动物中, 硬脑膜通过颈部肌肉的钝夹层暴露, 使套管插入到麦格纳 (CM) 的池中。套管由细斜面针或硼硅酸盐毛细管组成, 通过聚乙烯 (PE) 管连接到注射器。使用注射器泵, 分子可以直接注射在控制速率的 CM, 这是连续的蛛网膜下腔。从蛛网膜下腔, 我们可以通过对流流的周围空间来追踪脑脊液的通量, 从而产生与间质流体的溶质交换。CMc 可在手术后立即注射, 或用于慢性植入, 随后注射麻醉或清醒, 自由移动啮齿动物。定量的示踪剂分布在脑实质可以由荧光, 2 光子显微术, 磁共振成像 (MRI), 根据物理化学性质的注射分子。因此, CMc 结合各种成像技术, 为评估 glymphatic 系统和脑脊液动力学和功能提供了有力的工具。此外, CMc 可以作为一个管道, 快速, 大脑范围内传递的信号分子和代谢基质, 否则不能通过血脑屏障 (BBB)。

在立体定向的框架内固定小鼠或大鼠时, 枕顶区域周围的颈部肌肉被直接解剖, 以暴露出麦格纳 (厘米)。在小脑的尾端和髓质 (图 1A-1C) 之间, CM 的三角形结构容易被识别。套管通过轻轻刺穿寰枕膜 (图 1D), 插入到 CM 中 1-2 毫米。硬脑膜膜是一个强硬的结构和插入的套管是改善通过倾斜的动物头部的120°有关的身体。在注入泵的帮助下, 将不同标记的分子注入到麦格纳的池中以控制速率 (图 1E)。经过一段时间, 允许脑脊液示踪循环, 动物被安乐死。大脑是仔细解剖和固定的浸泡在4% 粉煤灰 o/n 在4摄氏度。从 CM 注射的啮齿目动物的大脑的宏观背观显示脑脊液示踪剂在小脑蛛网膜下储水池中的分布, 在嗅球和在大脑中动脉的 paravascular 空间 (MCAs) (图1F).在大脑的腹部, 宏观的观点显示, 沿威利斯 (图 1G) 的圆周上的脑脊液示踪物分布。CM 注入大脑的组织学切片进一步揭示了脑实质中示踪物的 paravascular 分布。麻醉下注射的小鼠 (图 1H) (或在自然睡眠期间, 见1) 显示, 与在他们的家庭笼中清醒和自由移动时注射的小鼠相比, 在脑实质中示踪剂分布显著增加 (图1I)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57378/57378fig1.jpg"> 图 1: 向麦格纳池中注入示踪剂.(A) 示意图概述了老鼠头和大脑, 显示了大池 (CM) 与大脑和颅骨结构的位置。(B)在周围的颈部肌肉被直率地解剖并推到两侧后, 显微照片暴露的 CM。(C) 在 B (黑色矩形) 中描述的区域的放大倍数, 显示厘米 (虚线) 的倒三角形结构及其与周围结构有关的位置,即枕嵴、小脑和髓质。(D)显微摄影插入到 CM. (E ) 方案中的鼠标固定头的侧面视图, 在与身体有关的120°角度略有倾斜。虚线矩形分隔区域的嵌入显示了一个旁视图方案的方案, 旁视图的鼠标大脑与套管插入到 CM, 如 E 所述。一个注射器, 这是耦合到注射泵, 是用来提供脑脊液示踪剂或造影药进入 CM 通过管连接到一个精细的30G 针。30分钟后, 用荧光示踪剂从背部的(F)和腹侧(G)方面观察到的一整只老鼠大脑的代表性图像。(H, I,)有代表性的冠状脑部分复染与 DAPI (4 ', 6-diamidino 2-苯基吲哚; 1 µg/毫升) 注射脑脊液示踪剂的小鼠被麻醉 (H) 和觉醒 (I), 30 分钟后, cm 注射的速度1µL/分钟5µLovalbumin-AF647 共轭 (OA, 45kDa; 2% aCSF) 和葡聚糖-FITC 共轭 (德克斯, 3kDa; 2% 在 aCSF) 的混合物的体积。刻度条, 5 毫米为 B, C, F, G;2毫米为 D, 和500µm 为 H, i. Cb, 小脑;厘米, 麦格纳池;Ctx, 皮质;还有嗅球<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57378/57378fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Cannula Implantation into the Cisterna Magna of Rodents at JoVE.com

Cannula Implantation into the Cisterna Magna of Rodents

将套管植入啮齿动物的麦格纳池中

在这里, 我们描述了一个协议, 以执行麦格纳插管 (CMc), 一种微创的方式, 提供示踪剂, 基质和信号分子进入脑脊液 (CSF)。结合不同的成像方式, CMc 使 glymphatic 系统和脑脊液动力学评估, 以及大脑范围内的各种化合物的交付。

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

cannula-implantation-into-the-cisterna-magna-of-rodents

Nanyang Technological University

Research

JoVE Journal

Neuroscience

43.9K 次观看.  University of Copenhagen. 在这里, 我们描述了一个协议, 以执行麦格纳插管 (CMc), 一种微创的方式, 提供示踪剂, 基质和信号分子进入脑脊液 (CSF)。结合不同的成像方式, CMc 使 glymphatic 系统和脑脊液动力学评估, 以及大脑范围内的各种化合物的交付。

视频: Cannula Implantation into the Cisterna Magna of Rodents

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

科研

神经科学

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.

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 ...

Direct Cannula Implantation in the Cisterna Magna of Pigs

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces and has been implicated in the clearance of neurotoxic peptides such as amyloid-beta. Impaired glymphatic function exacerbates disease pathology in animal models of neurodegenerative diseases, such as Alzheimer's, which highlights the importance of understanding this clearance system. The glymphatic system is often studied by cisterna magna cannulations (CMc), where tracers are delivered directly into the cerebrospinal fluid (CSF). Most studies, however, have been carried out in rodents. Here, we demonstrate an adaptation of the CMc technique in pigs. Using CMc in pigs, the glymphatic system can be studied at a high optical resolution in gyrencephalic brains and in doing so bridges the knowledge gap between rodent and human glymphatics.

This article presents a step-by-step protocol for the direct cannula implantation in the cisterna magna of pigs.

直接插管植入猪的蓄水池

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces ...

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. This standard approach can be challenging for targets in the caudal brainstem and upper cervical cord due to specific anatomical challenges as these sites are remote from skull landmarks, leading to imprecision. Here we outline an alternative stereotaxic approach via the cisterna magna that has been used to target discrete regions of interest in the caudal brainstem and upper cervical cord. The cisterna magna extends from the occipital bone to the atlas (i.e., the second vertebral bone), is filled with cerebrospinal fluid, and is covered by dura mater. This approach provides a reproducible route of access to select central nervous system (CNS) structures that are otherwise hard to reach due to anatomical barriers. Furthermore, it allows for direct visualization of brainstem landmarks in close proximity to the target sites, increasing accuracy when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. Finally, this approach provides an opportunity to avoid the cerebellum, which can be important for motor and sensorimotor studies.

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

立体定位手术方法 ，通过 大水池在小鼠中显微注射尾部脑干和上颈椎脊髓

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. ...

幼年大鼠腰椎穿刺 AAV9 递送用于 CNS 基因治疗

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

作者聚焦：评估幼年大鼠的鞘内基因治疗疗效

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

通过腰椎池注射在幼年大鼠中鞘内载体给药

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...

A Modified Method for Intrathecal Catheterization in Rats

Intrathecal catheterization has been widely applied in animal experiments, especially those on neuropathic pain. However, the traditional methods still have&#160;several limitations. Although some investigators have attempted to improve the traditional methods, the available methods still need to be modified. Herein, we introduce a modified method for intrathecal catheterization in rats.
This method uses a 20 cm long stainless-steel wire (0.2 mm in diameter), a 15 cm long plastic PE10 tube, a self-made sealing cap, and a 0.3 cm &#215; 0.5 cm anti-allergic band. Our modified method for intrathecal catheterization has several advantages. First, introducing a stainless-steel wire to PE10 tube increases the elasticity of the tube, improves the success rate of intrathecal catheterization, reduces the amount of space required for the operation, and minimizes the damage to the tissues around the lumbar spine. Second, the length of PE10 tube is determined before the surgery, and catheter indwelling time can be longer than one week. Third, the PE10 tube is fixed by a figure-8 suture, 4 times, which prevents tube movement and retraction when the animal moves. Fourth, a&#160;self-made sealing cap is used to seal the PE10 tube, which not only prevents cerebrospinal fluid leakage but also reduces the need for repeated cutting of PE10 tube. Finally, the extracorporeal end of PE10 tube is tied with a band, which prevents tube retraction when the animal moves.
This method can increase the catheterization success rate in rats, as approximately 80% of PE10 tubes remained in place even 28 days after surgery. Thus, this modified method may represent a simple, convenient, and reliable approach for repetitive intrathecal drug administration.

Herein, we introduce a modified method for intrathecal catheterization in rats that represents a simple, convenient, and reliable approach for repetitive intrathecal drug administration.

一种改良的大鼠鞘内导管插入术方法

Intrathecal catheterization has been widely applied in animal experiments, especially those on neuropathic pain. However, the traditional methods still ...

医学

Tracer Molecule Delivery in the Cisterna Magna: A Method to Deliver Fluorescent Tracer Molecules via Direct Cannulation of the Cisterna Magna in Pig

Source: B&#232;chet, N. B., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62641">Direct Cannula Implantation in the Cisterna Magna of Pigs.</a> J. Vis. Exp. (2021).
This video describes the protocol for tracer molecule delivery via direct cannulation of the cisterna magna in a pig brain. This method helps visualize the distribution of cerebrospinal fluid in the glymphatic system.

磁池中的示踪分子递送:一种通过猪磁池直接插管递送荧光示踪分子的方法

Source: B&#232;chet, N. B., et al. Direct Cannula Implantation in the Cisterna Magna of Pigs. J. Vis. Exp. (2021).
This video describes the protocol for ...

JoVE Encyclopedia of Experiments

生物学

Large Animal Models

Porcine

Surgical Cisterna Magna Injection: A Method for Administering Tumor Cells Directly into Central Nervous System of Murine Model

Source: Law, V. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62033/a-murine-ommaya-xenograft-model-to-study-direct-targeted-therapy">A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease.</a> J. Vis. Exp. (2021)
In this video, we describe the procedure to deliver human-derived tumor cells into the cisterna magna of a mouse&#8217;s brain. The orthotopic mouse model can be used to study metastatic spread of cancer to the cranial meninges.

手术 Cisterna Magna 注射液:一种将肿瘤细胞直接注入小鼠模型中枢神经系统的方法

Source: Law, V. et al. A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease. J. Vis. Exp. (2021)
In this video, we ...

将套管植入啮齿动物的麦格纳池中

摘要

探索更多视频

此视频中的章节

隔离啮齿类动物或人类的鼻嗅干细胞

通过宫内注射诱导利普托门宁皮细胞修饰

直接插管植入猪的蓄水池

立体定位手术方法，通过大水池在小鼠中显微注射尾部脑干和上颈椎脊髓

通过腰椎池注射在幼年大鼠中鞘内载体给药

通过腰椎池注射在幼年大鼠中鞘内载体给药

通过腰椎池注射在幼年大鼠中鞘内载体给药

一种改良的大鼠鞘内导管插入术方法

磁池中的示踪分子递送:一种通过猪磁池直接插管递送荧光示踪分子的方法

手术 Cisterna Magna 注射液:一种将肿瘤细胞直接注入小鼠模型中枢神经系统的方法

将套管植入啮齿动物的麦格纳池中

摘要

探索更多视频

此视频中的章节

隔离啮齿类动物或人类的鼻嗅干细胞

通过宫内注射诱导利普托门宁皮细胞修饰

直接插管植入猪的蓄水池

立体定位手术方法 ，通过 大水池在小鼠中显微注射尾部脑干和上颈椎脊髓

通过腰椎池注射在幼年大鼠中鞘内载体给药

通过腰椎池注射在幼年大鼠中鞘内载体给药

通过腰椎池注射在幼年大鼠中鞘内载体给药

一种改良的大鼠鞘内导管插入术方法

磁池中的示踪分子递送:一种通过猪磁池直接插管递送荧光示踪分子的方法

手术 Cisterna Magna 注射液:一种将肿瘤细胞直接注入小鼠模型中枢神经系统的方法

立体定位手术方法，通过大水池在小鼠中显微注射尾部脑干和上颈椎脊髓