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
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、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。
Protocol
Representative Results
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 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?…
Divulgations
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 |
References
- Tumani, H., Huss, A., Bachhuber, F. The cerebrospinal fluid and barriers – anatomic and physiologic considerations. Handbook of Clinical Neurology. , 21-32 (2017).
- Jessen, N. A., Munk, A. S., Lundgaard, I., Nedergaard, M. The Glymphatic System: A Beginner’s Guide. Neurochemical Research. 40 (12), 2583-2599 (2015).
- Iliff, J. J., et al. Impairment of glymphatic pathway function promotes tau pathology after traumatic brain injury. The Journal of Neuroscience. 34 (49), 16180-16193 (2014).
- Iliff, J. J., et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Science Translational Medicine. 4 (147), (2012).
- Louveau, A., et al. Structural and functional features of central nervous system lymphatic vessels. Nature. 523 (7560), 337-341 (2015).
- Peng, W., et al. Suppression of glymphatic fluid transport in a mouse model of Alzheimer’s disease. Neurobiology of Disease. 93, 215-225 (2016).
- Da Mesquita, ., S, , et al. Functional aspects of meningeal lymphatics in ageing and Alzheimer’s disease. Nature. 560 (7717), 185-191 (2018).
- Gaberel, T., et al. Impaired glymphatic perfusion after strokes revealed by contrast-enhanced MRI: a new target for fibrinolysis. Stroke. 45 (10), 3092-3096 (2014).
- Xavier, A. L. R., et al. Cannula Implantation into the Cisterna Magna of Rodents. Journal of Visualized Experiments. 10 (135), (2018).
- Plog, B. A., et al. Transcranial optical imaging reveals a pathway for optimizing the delivery of immunotherapeutics to the brain. JCI Insight. 3 (23), (2018).
- Kress, B. T., et al. Impairment of paravascular clearance pathways in the aging brain. Annals of Neurology. 76 (6), 845-861 (2014).
- Mestre, H., et al. Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension. Nature Communications. 9 (1), 4878 (2018).
- Xie, L., et al. Sleep drives metabolite clearance from the adult brain. Science. 342 (6156), 373-377 (2013).
- Plog, B. A., Nedergaard, M. The Glymphatic System. in Central Nervous System Health and Disease: Past, Present, and Future. Annual Review of Pathology. 13, 379-394 (2018).
- Iliff, J. J., et al. Brain-wide pathway for waste clearance captured by contrast-enhanced MRI. Journal of Clinical Investigation. 123 (3), 1299-1309 (2013).
- Davoodi-Bojd, E., et al. Modeling glymphatic system of the brain using MRI. Neuroimage. 188, 616-627 (2019).
- Lee, H., et al. The Effect of Body Posture on Brain Glymphatic Transport. The Journal of Neuroscience. 35 (31), 11034-11044 (2015).
- Hablitz, L. M., et al. Increased glymphatic influx is correlated with high EEG delta power and low heart rate in mice under anesthesia. Science Advances. 5 (2), (2019).
- Rasmussen, M. K., Mestre, H., Nedergaard, M. The glymphatic pathway in neurological disorders. The Lancet Neurology. 17 (11), 1016-1024 (2018).
- Mestre, H., et al. Aquaporin-4-dependent glymphatic solute transport in the rodent brain. Elife. 7, (2018).
- Monai, H., et al. Calcium imaging reveals glial involvement in transcranial direct current stimulation-induced plasticity in mouse brain. Nature Communications. 7, 11100 (2016).
- Munk, A. S., et al. PDGF-B Is Required for Development of the Glymphatic System. Cell Reports. 26 (11), 2955-2969 (2019).
- Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nature Methods. 9 (7), 676-682 (2012).
- Ren, Z., et al. Hit & Run’ model of closed-skull traumatic brain injury (TBI) reveals complex patterns of post-traumatic AQP4 dysregulation. Journal of Cerebral Blood Flow & Metabolism. 33 (6), 834-845 (2013).
- Plog, B. A., et al. Biomarkers of traumatic injury are transported from brain to blood via the glymphatic system. The Journal of Neuroscience. 35 (2), 518-526 (2015).
- Ma, Q., Ineichen, B. V., Detmar, M., Proulx, S. T. Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice. Nature Communications. 8 (1), 1434 (2017).
- Roth, T. L., et al. Transcranial amelioration of inflammation and cell death after brain injury. Nature. 505 (7482), 223-228 (2014).
- Xu, H. T., Pan, F., Yang, G., Gan, W. B. Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nature Neuroscience. 10 (5), 549-551 (2007).
- Ma, Q., et al. Rapid lymphatic efflux limits cerebrospinal fluid flow to the brain. Acta Neuropathologica. 137 (1), 151-165 (2019).
- Silasi, G., Xiao, D., Vanni, M. P., Chen, A. C., Murphy, T. H. Intact skull chronic windows for mesoscopic wide-field imaging in awake mice. Journal of Neuroscience Methods. 267, 141-149 (2016).
