相干反斯托克斯拉曼光谱（CARS）在脑切片中成像髓鞘形成的应用
Summary
可视化髓鞘形成是许多研究神经系统的研究人员的重要目标。CARS是一种与免疫荧光兼容的技术，可以对组织内的脂质进行天然成像，例如大脑，照亮髓磷脂等特殊结构。
Abstract
相干反斯托克斯拉曼光谱（CARS）是化学家和物理学家经典采用的一种技术，用于产生分子特征振动的相干信号。然而，这些振动特征也是解剖组织（如大脑）内分子的特征，使其越来越有用和适用于神经科学应用。例如，CARS可以通过特异性激发这些分子内的化学键来测量脂质，从而可以量化组织的不同方面，例如参与神经传递的髓磷脂。此外，与通常用于定量髓磷脂的其他技术相比，CARS还可以设置为与免疫荧光技术兼容，允许与其他标记物（如钠通道或突触传递的其他成分）共同标记。髓鞘形成变化是脱髓鞘疾病（如多发性硬化症）或其他神经系统疾病（如脆性X综合征或自闭症谱系障碍）的固有重要机制，是一个新兴的研究领域。总之，CARS可以以创新的方式用于回答神经科学中的紧迫问题，并为与许多不同神经系统疾病相关的潜在机制提供证据。
Introduction
动作电位是大脑中信息的基本单位，通过轴突传播的动作电位构成了信息处理的支柱1，2，3。神经元通常接收来自多个其他神经元的传入输入，并在给定的狭窄时间窗口4，5内整合这些输入。因此，轴突中作用电位传播的机制受到了研究者的极大关注。
当通过轴突传播时，动作电位沿轴突反复再生，以确保可靠的传播6。在颌脊椎动物（gnathostomes）的大多数神经元中，轴突被髓磷脂鞘包围，髓鞘是由附近的少突胶质细胞或雪旺细胞产生的富含脂质的物质，这是神经胶质细胞的类型（回顾于7，8）。这种髓鞘对轴突进行电绝缘，降低其电容，并允许动作电位高效、快速且能耗更低。髓磷脂不能均匀地覆盖轴突，但它将轴突包裹在它们之间有短间隙的段中，称为Ranvier的节点（在9，10中回顾）。控制轴突电绝缘水平的髓鞘厚度和控制动作电位沿轴突再生频率的Ranvier节点间距都会影响动作电位传播的速度（在11中回顾）。
有大量文献表明髓鞘厚度影响轴突12，13，14中动作电位传播的速度。此外，轴突髓鞘形成的改变可导致许多CNS缺陷15，16，17，18，19，20，21。因此，许多研究工作的重点涉及轴突髓鞘形成的测量和表征也就不足为奇了。髓鞘厚度的测量最常使用电子显微镜进行，这种技术需要大量的组织准备，并且与免疫组织化学结合使用具有挑战性。然而，还有一种基于相干反斯托克斯拉曼光谱（CARS）的更快、更简单的轴突髓鞘形成测量技术。CARS激光器可以调谐到各种频率，当调谐到适合激发脂质的频率时，髓磷脂可以成像，而无需任何额外的标记22。脂质成像可以与标准免疫组织化学相结合，使得脂质可以与几个荧光通道一起成像23。使用CARS对髓鞘形成进行成像明显快于电子显微镜，并且其分辨率尽管低于EM，但足以检测相同类型轴突中髓鞘形成的微小差异。
Protocol
所有实验均符合所有适用法律、美国国立卫生研究院指南，并已获得科罗拉多大学安舒茨机构动物护理和使用委员会的批准。 1. 动物 使用从杰克逊实验室获得的C57BL / 6J（股票#000664）小鼠（Mus musculus ）或最初从查尔斯河获得的蒙古沙鼠（Meriones unguiculatus）。 2. 组织准备 对于经心灌注，用戊巴比?…
Representative Results
与其他技术相比，CARS显微镜的最大优势之一是与荧光成像的兼容性23。 图1 显示了CARS光谱与用免疫荧光标记标记的Nissl相比，光谱几乎没有重叠。 图2 显示了结合共聚焦显微镜的CARS激光设置。 图3 显示了两个代表性图像，一个是单个堆栈，另一个是来自沙鼠和小鼠的z-stack最大投影，可以使用显示细胞体（青色?…
Discussion
越来越多的文献强调髓磷脂在大脑功能中的作用13，16，21，28。此外，我们知道髓鞘厚度和髓鞘形成模式可以在多种神经系统疾病中发生变化，例如多发性硬化症（29 年回顾）、衰老（30 年回顾）、自闭症20、31 等。因此?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
由 NIH R01 DC 17924、R01 DC 18401 （Klug） 和 NIH 1R15HD105231-01、T32DC012280 和 FRAXA （McCullagh） 提供支持。CARS成像是在科罗拉多大学安舒茨医学院神经技术中心的先进光学显微镜核心部分进行的，部分由NIH P30 NS048154和NIH P30 DK116073支持。
Materials
| Anesthetic: | |||
| 1 mL disposable syringe with needle 27 GA x 0.5" | Exel int | 260040 | |
| Fatal + | Vortech | ||
| Surgery: | |||
| Spring Scissors – 8mm Cutting Edge | Fine Science Tools | 15024-10 | |
| Standard tweezers | Fine Science Tools | 11027-12 | |
| Perfusion: | |||
| 4% Paraformaldehyde | Fisher Chemical | SF994 (CS) | |
| Fine Scissors – Sharp | Fine Science Tools | 14063-11 | |
| Kelly hemostats | Fine Science Tools | 13019-14 | |
| Millipore H2O | |||
| Needle tip, 23 GA x 1" | BD precision glide | 305193 | |
| Phosphate buffered saline (PBS): | |||
| Potassium chloride | Sigma | P9333 | |
| Potassium phosphate monobase | Sigma | P5655 | |
| pump with variable flow or equivalent | |||
| Sodium chloride | Fisher Chemical | s271-1 | |
| Sodiumphosphate dibasic | Sigma | S7907 | |
| Dissection: | |||
| 50 mL vial with 4% PFA | |||
| Bochem Chemical Spoon 180mm | Bochem | 230331000 | |
| Fine Scissors – Sharp | Fine Science Tools | 14063-11 | |
| Noyes Spring Scissors | Fine Science Tools | 15011-12 | |
| Pair of fine (Graefe) tweezers | Fine Science Tools | 11050-10 | |
| Shallow glass or plastic tray, approximately 10" x 10" | |||
| Standard tweezers | Fine Science Tools | 11027-12 | |
| Surgical Scissors – Blunt | Fine Science Tools | 14000-20 | |
| Slicing: | |||
| Agar, plant | RPI | 9002-18-0 | |
| Vibratome | Leica | VT1000s | |
| well plate | Alkali Sci. | TPN1048-NT | |
| Staining: | |||
| AB Media: | 1n 1,000 mL of Millipore H2O | ||
| Phosphate buffered (PB): | |||
| Potassium Phosphate Monobase | Sigma | P5655 | |
| Sodium Phosohate Dibasic | Sigma | S7907 | |
| BSA (Bovine serum albumin) | Sigma life science | A2153-100g | |
| Sodium Chloride | Fisher Chemical | s271-1 | |
| Triton X-100 | Sigma – Aldrich | x100-500ml | |
| Nissl 435/455 | Invitrogen | N21479 | |
| CARS: | |||
| APE picoemerald laser | Angewandte Physik & Elektronik GmbH | ||
| bandpass filter (420-520 nm) | Chroma Technology | HQ470/100m-2P | |
| bandpass filter (500-530 nm) | Chroma Technology | HQ515/30m-2P | |
| bandpass filters (640-680 nm) | Chroma Technology | HQ660/40m-2P | |
| Confocal microscope | Olympus | FV1000 | |
| Cut Transfer pipet | Fisher | 13-711-7M | |
| dichroic longpass 565 nm | Chroma Technology | 565dcxr | |
| dichroic longpass 585 nm | Chroma Technology | 585dcxr | |
| dichroic shortpass 750 nm | Chroma Technology | T750spxrxt | |
| glass bottom culture dish | MatTek | P35G-0-10-C | |
| glass weight (10 mm x 10 mm boro rod) | Allen Scientific Glass Inc | ||
| multiphoton shortpass emission filter 680 nm | Chroma Technology | ET680sp-2p8 | |
| PBS |
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Citazione di questo articolo
McCullagh, E. A., Poleg, S., Stich, D., Moldovan, R., Klug, A. Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices. J. Vis. Exp. (185), e64013, doi:10.3791/64013 (2022).