Summary

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

Published: July 29, 2019
doi:

Summary

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。

Abstract

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。

Introduction

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、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。

Protocol

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 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。 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?…

Representative Results

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?…

Discussion

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 3H-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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

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Cite This Article
Sweeney, A. M., Plá, V., Du, T., Liu, G., Sun, Q., Peng, S., Plog, B. A., Kress, B. T., Wang, X., Mestre, H., Nedergaard, M. In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy. J. Vis. Exp. (149), e59774, doi:10.3791/59774 (2019).

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