Name: in vivo imaging of cerebrospinal fluid transport through the intact mouse skull using fluorescence macroscopy
Uploaded: 2019-07-29T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy at JoVE.com

这项工作由美国国家神经疾病和中风研究所和国家老龄问题研究所(美国国家卫生研究院;美国国家卫生研究院;美国国家卫生研究院;美国国家卫生研究院;美国国家卫生研究院;美国国家卫生研究院;美国国家卫生研究院;美国国家卫生研究院;美国国家卫生研究院;美国国家卫生研究院;美国国家卫生研究院;美国国家卫生研究院;美国国家卫生研究院;美国国家R01NS100366 和 RF1AG057575 至 MN、基金会 Leducq 跨大西洋卓越网络计划以及欧盟 Horizon 2020 研究和创新计划(批号 666881;SVD_目标)。我们还要感谢薛丹在图形插图方面的专家协助。

所有实验均获得罗切斯特大学动物资源委员会(UCAR,第2011-023号议定书)的批准,并根据NIH《实验室动物护理和使用指南》进行。
1. 准备蓄水池、头板和头架
<ol><li>手术前对所有手术器械和头板进行消毒。 注:荧光示踪剂通过蓄水池直接输送到CSF。有关本程序的详细说明,请参阅 Xavier 等人9。</li>
	<li>简单地说,使用针驱动器,将 30G x 12.7 mm(1/2 英寸)针头的尖端打?...

<ol>
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我们描述了使用市售荧光宏镜和示踪剂在活小鼠中执行颅内CSF成像的详细方案。这种技术是简单和微创的,但定量的。体内成像与敏感方法(如无线电标记示踪剂的液体闪烁计数)(包括 CM 交付后 3个H-dextran 和14C-inulin)以及外部活晕节定量10相关, 18.用双光子显微镜验证表明,在宏观镜下皮质血管上看到的CSF示踪剂主要位于MCA及其?...

脑脊液(CSF)沐浴大脑和脊髓,并参与维持平衡,提供营养,调节颅内压力1。在亚阿拉奇诺伊空间的 CSF 通过周围皮动脉的周围血管空间 ( PVS ) 网络进入大脑 , 然后沿着穿透性动脉 2向动。一旦进入脑膜,CSF与间质流体(ISF)交换,携带有害的代谢物,如淀粉样蛋白-β(A+)和tau蛋白聚集于大脑,通过低电阻白质道和渗透空间2,3.这种途径依赖于星体水孢素-4(AQP4)通道,因此被称为胶质淋巴(淋巴)系统4。神经桩的废物最终通过颅神经附近的淋巴血管和脑向宫颈淋巴结5清除从CSF-ISF。这个系统的失败与多种神经疾病有关,如阿尔茨海默氏病6、7、创伤性脑损伤3、缺血和出血性中风8。
CSF运输可以通过注入示踪剂到蓄水池(CM)9,10和淋巴研究,在过去主要使用双光子显微镜4,11,12, 13、磁共振成像(MRI)14、15、16、17、前体成像3、6、11、18以评估示踪动力学。双光子显微镜是一种合适的方法,用于在PVS和parenchyma中对CSF示踪剂进行详细成像,由于其空间分辨率高,但是,它有一个狭窄的视野,需要侵入性颅窗口或颅骨变薄。外生成像,结合免疫组织化学,可实现从单个细胞到整个大脑19的多层次分析。然而,观察验尸组织所需的灌注固定过程在CSF流动方向上产生深刻变化,使PVS崩溃,显著改变示踪剂12的分布和位置。最后,虽然MRI可以跟踪整个鼠和人脑的CSF流,但它缺乏血管外流动的空间和时间分辨率。
一项新技术,即颅内宏观成像,通过在活小鼠的整个背皮层中实现血管外脑CSF传输的广域成像,解决了其中一些局限性。这种类型的成像是使用多波段滤波器立方体、可调LED光源和高效CMOS摄像机10的荧光宏镜完成的。这些设置能够解决PVS高达1-2毫米以下的头骨表面,并可以检测荧光团高达5-6毫米以下的皮质表面,同时保持头骨完全完整10。能够快速调整激发波长的多波段滤波器和 LED 能够使用多个荧光团,使 CSF 在同一实验中具有不同分子量和化学特性的示踪剂。
这个过程需要一个简单的,微创手术,以暴露头骨和放置一个轻量级的头板,以稳定头部在成像过程中。追踪器可以交付到CM,而无需钻入头骨或穿透皮质组织与移液器或管9,20。与传统的端点可视化相比,CM 管和头板在数天到数周内保持稳定,便于进行更复杂的实验设计。该协议描述了在急性或慢性荧光CSF示踪剂注射到麻醉/睡眠或清醒小鼠的CM中后,如何利用颅内宏观成像来研究淋巴系统功能。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>0.25% Bupivacaine HCl</td>
			<td></td>
			<td></td>
			<td>University of Rochester Vivarium</td>
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			<td>100 &#181;L Gastight Syringe Model 1710 TLL, PTFE Luer Lock</td>
			<td>Hamilton Company</td>
			<td>81020</td>
			<td></td>
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			<td>A-M Systems Dental Cement Powder</td>
			<td>Fisher Scientific</td>
			<td>NC9991371</td>
			<td></td>
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			<td>Carprofen</td>
			<td></td>
			<td></td>
			<td>University of Rochester Vivarium</td>
		</tr>
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			<td>Chlorhexidine</td>
			<td>Prevantics</td>
			<td>B10800</td>
			<td></td>
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			<td>CMOS Camera</td>
			<td>Hammamatsu</td>
			<td>ORCA Flash 4.0</td>
			<td></td>
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			<td>Head Plate</td>
			<td>University of Rochester</td>
			<td>No catalog #</td>
			<td>Custom made at the machine shop at the University of Rochester</td>
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			<td>High-Temperature Cautery</td>
			<td>Bovie Medical Corporation</td>
			<td>AA01</td>
			<td></td>
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			<td>Insta-set Accelerator</td>
			<td>Bob Smith Industries</td>
			<td>BSI-151</td>
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			<td>Isoflurane - Fluriso</td>
			<td>Vet One</td>
			<td>502017</td>
			<td>University of Rochester Vivarium</td>
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			<td>Ketamine</td>
			<td></td>
			<td></td>
			<td>Strong Memorial Hospital Pharmacy</td>
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			<td>Krazy Glue</td>
			<td>Elmer&#39;s Products, Inc</td>
			<td>No catalog #, see link in comments</td>
			<td>https://www.amazon.com/Krazy-Glue-KG48348MR-Advance-Multicolor/dp/B000BKO6DG</td>
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			<td>Micropore Surgical tape</td>
			<td>Fisher Scientific</td>
			<td>19-027-761</td>
			<td></td>
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			<td>Paraformaldehyde</td>
			<td>Sigma-aldrich</td>
			<td>P6148</td>
			<td></td>
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			<td>PE10 - Polyethylene .011&#34; x .024&#34; per ft., 100 ft. continuous</td>
			<td>Braintree Scientific</td>
			<td>PE10 100 FT</td>
			<td></td>
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			<td>Pump 11 Elite Infusion Only Dual Syringe</td>
			<td>Harvard Apparatus</td>
			<td>70-4501</td>
			<td></td>
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			<td>PURALUBE VET OINTMENT</td>
			<td>Dechra</td>
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			<td>Puritan PurSwab Cotton Tipped Cleaning Sticks</td>
			<td>Fisher Scientific</td>
			<td>22-029-553</td>
			<td></td>
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			<td>Research Macro Zoom Microscope</td>
			<td>Olympus</td>
			<td>MVX10</td>
			<td></td>
		</tr>
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			<td>Simple Head Holder Plate (for mice)</td>
			<td>Narishige International USA Inc</td>
			<td>MAG-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-use Needles, BD Medical</td>
			<td>VWR</td>
			<td>BD305106</td>
			<td></td>
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			<td>Sterile Alcohol Prep Pads</td>
			<td>Fisher Scientific</td>
			<td>22-363-750</td>
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			<td>Tunable LED</td>
			<td>PRIOR Lumen 1600-LED</td>
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			<td></td>
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			<td>Xylazine</td>
			<td></td>
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			<td>University of Rochester Vivarium</td>
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	</tbody>
</table>

in vivo imaging of cerebrospinal fluid transport through the intact mouse skull using fluorescence macroscopy

啮齿动物的脑脊液(CSF)流动主要使用示踪剂的活体定量进行研究。双光子显微镜和磁共振成像(MRI)等技术在体内实现了CSF流量的定量,但分别受到成像体积减少和空间分辨率低的限制。最近的研究发现,CSF通过围绕啮齿动物皮层的皮层和穿透动脉周围的血管空间网络进入脑血管瘤。CSF的这种血管外进入是淋巴系统的主要驱动力,这是一种与清除有毒代谢溶质(例如淀粉样蛋白-β)有关的途径。在这里,我们演示了一种新的宏观成像技术,它允许通过活小鼠的完整头骨对荧光CSF示踪剂进行实时、中观成像。这种微创方法有助于多种实验设计,并支持对CSF动力学进行单次或重复测试。宏镜具有高空间和时间分辨率,其较大的龙门和工作距离允许在行为设备上执行任务时进行成像。这种成像方法已通过从该技术获得的双光子成像和荧光测量与外源荧光和无线电标记示踪剂的定量密切相关。在此协议中,我们描述了如何使用颅内宏观成像来评估活小鼠的淋巴迁移,为更昂贵的成像模式提供了一种可获取的替代方法。

CSF的流入在表显荧光宏观镜上成像(图1A),它允许在鼠皮层中对CSF示踪传输进行中显成像。整个头骨头板允许可视化的鼻骨,正面和前部骨骼在中心,和骨骼的间骨节的玫瑰部分(图1B)。在成像过程中,鼻腔、下垂、冠状和羔羊丝缝合线很容易识别(图1C)。一旦CSF示踪剂开始注入CM(图1D),示踪荧光首先出现在基底蓄水?...

Watch this Scientific Journal Video about In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy at JoVE.com

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

颅内光学成像允许通过完整的头骨在活小鼠皮层中对脑脊液进行宽场成像。

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 ...

这项新技术用于实时中观成像荧光CSF痕迹通过活鼠的完整头骨可用于评估淋巴运输。主要优点是，这种技术能够以其他成像方式的一小部分成本对体内内颅内示踪剂进行动态测量。将头板放在 cisterna 磁石管上所需的程序简单且具有微创性，但需要正确操作。需要直观地演示此方法的关键步骤，以最大限度地提高图像质量，并实现不同小鼠和实验组之间的可重复比较。在确认脚趾捏湿颈部和头部与无菌水，并剃湿的毛皮反应不足后。用酒精拭子擦拭裸露的皮肤，去除任何残留的头发，将鼠标放在温度控制垫顶部的立体定向框架中。将软膏涂抹在动物的眼睛上，用氯环己丁拭子清洁裸露的皮肤。2分钟后，用酒精擦拭去除氯己丁，并涂抹碘溶液。当iodone干燥注射皮下镇痛到头骨和颈部的顶部。从覆盖腹皮的颈部部分开始，在覆盖的皮肤上进行中线切割，继续向轨道内线持续。横向将切口延伸至边界，其中时间肌肉插入头骨，并去除模糊切口的所有皮肤，以暴露正面骨骼和面骨。用无菌盐水灌溉头骨，并使用棉签清洁表面，直到没有碎屑和头发。插入西斯特纳磁石罐后，将牙科水泥和氰丙烯酸酯混合物涂抹在边界周围头板的腹侧。将头板放在头骨上，使板的前部边框与鼻骨的后尖端对齐，后边框与侧骨的前部对齐，确保下垂缝合线相对于窗口居中并直。请务必确保头板已固定，并且不会妨碍 Cisterna 磁石罐的视场。使用几滴胶水加速器固定头板的位置，用水泥混合物填充所有剩余的间隙。将 cisterna 磁格纳罐粘在头板上，并使用头板将鼠标放入固定位置的头部支架中。小心地将鼠标和连接到管上的输液泵放在推车上，以运送到宏观示波器上，然后将头架放在宏示波器的舞台上。确保从注射器泵到 cisterna 磁石管的管路没有紧绷，并且没有扭结。观察粘膜的呼吸速率和粉红色着色，以确认良好的氧合。打开宏镜摄像头和发光二极管，然后启动实时模式。调整成像场的放大倍率，使场顶部的鼻前缝合线和底部的羊皮缝合线可以清晰可视化。一旦将头部支架固定到大内窥镜阶段，将宏观镜聚焦在暴露的头骨上，直到焦平面位于日冕缝合线后部骨骼的侧侧。对于 CSF 示踪剂输液，将输液泵设置为适当的速率和体积，并设置每个通道的示踪剂的适当激励波长和曝光时间。然后，在同时启动示踪剂输液和成像之前，检查宏镜上的触发功能是否正确。在成像过程中，鼻索前体、下垂、日冕和羊皮素缝合线很容易识别。一旦CSF示踪剂被注入西斯特纳磁石，示踪剂荧光首先在大型的亚布拉奇内德CSF池中观察到，在奥菲托前蓄水池和松树凹槽附近的四分米蓄水池，并最终围绕中脑动脉。然后，CSF 示踪器沿着中脑动脉皮质皮皮分的血管空间进入大脑。创伤性脑损伤后CSF运输成像显示示踪剂最初在奥菲多前体蓄水池，但沿着皮质周围血管空间的淋巴流入在损伤的一侧被完全废除。体内图像的定量分析表明，与反边半球相比，伊皮拉特流入面积减少了近三分之一。为了避免运动伪影和焦平面的变化，请记住正确固定头板和头板支架，并检查动物的麻醉水平。实验后，通过使用实际的该技术量化示踪剂穿透率，可以进一步验证实验后，以生理和微创的方式研究淋巴系统，并可以帮助解决未来有关CSF在健康和疾病中流体动力学的问题。

Research

JoVE Journal

Neuroscience

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

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

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

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 ...

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

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 ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

人脑脊液 Endopeptidomic 分析的样品制备

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

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 ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

切除小鼠神经神经纤维神经丝的成像与分析

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...