使用路西法黄电磷学可视化星形形态
Summary
星形细胞是形态复杂的细胞，其多重过程和茂密的区域就是例证。为了分析它们的精细形态，我们提出了一种可靠的方案，用于在轻度固定组织中执行细胞内路西法黄色淋病。
Abstract
星形细胞是神经回路的重要组成部分。它们将整个中枢神经系统 （CNS） 分片，并参与各种功能，包括神经递质清除、组子调节、突触调节、神经元代谢支持和血流调节。星形细胞是一个复杂的细胞，具有索马，几个主要分支，和许多精细的过程，接触神经皮内不同的细胞元素。为了评估星形细胞的形态，有必要有一个可靠和可重复的方法来可视化其结构。我们报告一种可靠的方案，在成年小鼠的轻度固定脑组织中使用荧光路西法黄色（LY）染料对星形细胞进行细胞内电泳。该方法具有几个特征，可用于表征星形细胞形态。它允许对单个星形细胞进行三维重建，这有利于对其结构的不同方面进行形态分析。免疫组织化学与LY电泳也可用于了解星形细胞与神经系统不同成分的相互作用，并评估标记的星形细胞中蛋白质的表达。该协议可以在各种中枢神经系统疾病小鼠模型中实现，用光显微镜严格检查星形细胞形态。LY风泳提供了一种评估星形细胞结构的实验方法，特别是在损伤或疾病的情况下，这些细胞被建议经历显著的形态变化。
Introduction
星形细胞是中枢神经系统（CNS）中最丰富的胶质细胞。它们在ion平衡、血流调节、突触形成以及消除和神经递质接受1中发挥作用。星形细胞函数的范围广泛反映在其复杂的形态结构2，3。星形细胞包含几个初级和次要分支，它们分为数千个细枝和传单，直接与突触、树突、斧头、血管和其他胶质细胞相互作用。星形细胞形态因大脑区域而异，这可能暗示它们在神经元回路4中不同地执行其功能的能力。此外，星形细胞已知在发育期间、生理条件和多种疾病状态3、5、6期间改变其形态。
需要一种一致、可重复的方法来准确解决星形细胞形态的复杂性。传统上，免疫性细胞化学已经被用来可视化星形细胞与星形细胞特定或星形细胞富集蛋白质标记。然而，这些方法揭示了蛋白质表达的模式，而不是星形细胞的结构。常用的标记物，如胶质纤维酸性蛋白（GFAP）和S100钙结合蛋白β（S100+），不表达在整个细胞体积中，因此不解决完整的形态7。在星形细胞中普遍存在的荧光蛋白（病毒注射或转基因小鼠报告线）的遗传方法可以识别更细的分支和整体区域。然而，很难区分单个星形细胞，并且分析可能偏向于特定启动子8所针对的星形细胞群。串行截面电子显微镜已被用来揭示星形细胞过程与突触相互作用的详细图片。由于数以千计的星形细胞过程接触突触，目前无法用这种技术9重建整个细胞，尽管随着使用机器学习方法进行数据分析，这种情况预计会发生变化。
在本报告中，我们重点介绍了使用细胞内风泳与Lucifer黄色（LY）染料来描述小鼠星形细胞的过程，以CA1层辐射为例。该方法是基于埃里克·布松和马克·埃利斯曼10、11的开创性过去工作。从轻固定脑切片的星形细胞由其独特的索马形状识别，并充满LY。然后，细胞通过共聚焦显微镜进行成像。我们演示了如何使用LY淋巴磷光来重建单个星形细胞，并对其过程和领地进行详细的形态分析。此外，该方法可与免疫组织化学结合使用，以识别星形细胞和神经元、其他胶质细胞和脑血管之间的空间关系和相互作用。我们认为LY电泳是一个非常合适的工具，用于分析不同大脑区域的形态和小鼠模型的健康或疾病状况7，12，13。
Protocol
这项研究中的动物实验是按照美国国家卫生指南，在实验室动物的护理和使用，并批准校长的动物研究委员会在洛杉矶加州大学。所有实验中都使用了混合性别的成年小鼠（6-8周大）。 1. 解决方案准备 人工脑脊液（ACSF）溶液 在每个实验前制备新鲜的 ACSF 溶液（135 mM NaCl、5 mM KCl、1 mM MgCl 2、14.7 mM NaHCO3、11mM D-葡萄糖、1.25 mM Na2HPO<su…
Representative Results
这项研究中报告的数据来自每个实验中4只小鼠的7-12个细胞。在适当情况下，在图形面板中报告平均数据。 为了评估星形细胞形态，我们使用LY染料进行细胞内风泳，以填充CA1层辐射中的星形细胞，如图1总结。图2描绘了一个代表性的星形细胞及其精细的形态结构。使用 488 nm 激光线（步长 0.3 μm 和 3.0±3.5 倍数字变焦）在共聚焦激光扫描显微…
Discussion
本文概述的方法描述了一种在轻度固定脑切片中使用LY染料细胞内电磷化来可视化星形细胞形态的方法。该协议中强调了几个关键因素，有助于成功的LY淋巴磷酸和细胞的形态重建。一个因素是图像的质量和可重复性，这主要取决于小鼠的年龄和灌注的结果。在这项研究中，我们使用6~8周大的C57/BL6N小鼠。成功的灌注（高度依赖于渗透针的正确放置，并注意到由白色的大脑和脑血管中没有血液）是?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢索托女士、余博士和Octeau博士对文本的指导以及评论。NS060677 支持此工作。
Materials
| 10% Buffered Formalin Phosphate | Fisher | SF 100-20 | An identical alternative can be used |
| Acrodisc Syringe Filters with Supor Membrane | Pall | 4692 | An identical alternative can be used |
| Ag/AgCl ground pellet | WPI | EP2 | A similar alternative can be used |
| Alexa Fluor 546 goat anti-chicken IgG (H+L) | Thermo Scientific | A-11040 | A similar alternative can be used |
| Alexa Fluor 647 goat anti-rabbit IgG (H+L) | Thermo Scientific | A27040 | A similar alternative can be used |
| Anti Aquaporin-4 antibody | Novus Biologicals | NBP1-87679 | A similar alternative can be used |
| Anti GFAP antibody | Abcam | ab4674 | A similar alternative can be used |
| Borosilicate glass pipettes with filament | World precision instruments | 1B150F-4 | |
| C57BL/6NTac mice | Taconic Stock | B6 | A similar alternative can be used |
| Calcium Chloride | Sigma | 21108 | An identical alternative can be used |
| Confocal laser-scanning microscope | Olympus | FV1000MPE | A similar alternative can be used |
| D-glucose | Sigma | G7528 | An identical alternative can be used |
| Disodium Phosphate | Sigma | 255793 | An identical alternative can be used |
| Electrode puller- Model P-97 | Sutter | P-97 | A similar alternative can be used |
| Fluoromount-G | Southern Biotech | 0100-01 | An identical alternative can be used |
| Heparin sodium injection (1,000 USP per mL) | Sagent Pharmaceuticals | 400-10 | An identical alternative can be used |
| Imaris software (Version 7.6.5) | Bitplane Inc. | A similar alternative can be used | |
| Isofluorane | Henry Schein Animal Health | 29404 | An identical alternative can be used |
| Lidocaine Hydrochloride Injectable (2%) | Clipper | 1050035 | An identical alternative can be used |
| Lucifer Yellow CH dilithium salt | Sigma | L0259 | |
| Lucifer Yellow CH dipotassium salt | Sigma | L0144 | |
| Magnesium Chloride | Sigma | M8266 | An identical alternative can be used |
| Microscope Cover Glass | Thermo Scientific | 24X60-1 | An identical alternative can be used |
| Microscope Slides | Fisher | 12-544-2 | An identical alternative can be used |
| Normal Goat Serum | Vector Laboratories | S-1000 | An identical alternative can be used |
| Objective lens (40x) | Olympus | LUMPLFLN 40XW | A similar alternative can be used |
| Objective lens (60x) | Olympus | PlanAPO 60X | A similar alternative can be used |
| PBS tablets, 100 mL | VWR | VWRVE404 | An identical alternative can be used |
| Pipette micromanipulator- Model ROE-200 | Sutter | MP-285 / ROE-200 / MPC-200 | A similar alternative can be used |
| Potassium Chloride | Sigma | P3911 | An identical alternative can be used |
| Sodium Bicarbonate | Sigma | S5761 | An identical alternative can be used |
| Sodium Chloride | Sigma | S5886 | An identical alternative can be used |
| Stimulator- Model Omnical 2010 | World precision instruments | Omnical 2010 | A similar alternative can be used |
| Triton X 100 | Sigma | T8787 | An identical alternative can be used |
| Vibratome- Model #3000 | Pelco | 100-S | A similar alternative can be used |
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Cite This Article
Moye, S. L., Diaz-Castro, B., Gangwani, M. R., Khakh, B. S. Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis. J. Vis. Exp. (151), e60225, doi:10.3791/60225 (2019).