评估视网膜小胶质细胞吞噬功能<em>在体内</em>使用基于流式细胞术流量测定
Summary
在组织的动态平衡和吞噬功能不足的维护工作在病理上被牵连的小胶质细胞的吞噬作用是至关重要的。然而， 在体内评估小胶质细胞的功能在技术上具有挑战性。我们已经开发出一种简单而坚固的技术用于精确监测和量化小胶质细胞的吞噬潜力在生理环境。
Abstract
Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer’s and age-related macular degeneration, where amyloid-β plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.
Introduction
该方法的总的目标是要准确评估和体内小胶质细胞吞噬量化。小胶质细胞是中枢神经系统（CNS）的组织驻留的巨噬细胞。它们执行各种功能，以确保组织稳态的维持。这些包括免疫监视，神经营养因子的分泌，并为至关重要，吞噬作用1。小胶质细胞的吞噬作用是在诸如无关突触（突触修剪）和去除神经元凋亡2-4的吞噬大脑和视网膜的发育过程中的几个重要事件的关键。此外，损坏或凋亡细胞，细胞碎片和入侵微生物的小胶质细胞的吞噬作用已被证明是通过成年5维持CNS动态平衡是至关重要的。最后，小胶质细胞的吞噬作用已牵涉几种神经变性疾病，包括阿尔茨海默病和与年龄有关的发病黄斑变性，它已被认为有缺陷或不足的吞噬能力可能有助于淀粉样蛋白β（Aβ）斑块和玻璃疣，分别6,7的积聚。
小胶质细胞功能紧密可以通过微调节，特别是通过可溶性因子如肿瘤生长因子β或细胞 – 细胞相互作用。神经元组成型表达几个细胞表面配体，例如CD200和CX3CL1，而小胶质细胞只表达相应受体CD200R和CX3CR1。这些受体包含在它们的细胞内部分基于免疫受体酪氨酸的抑制基序（ITIMs）。这些抑制剂受体是用于防止小胶质细胞的过度刺激，从而可以向神经炎症的关键。因此，在正常生理条件下，神经元和小胶质细胞之间的细胞 – 细胞相互作用保持小胶质细胞处于静止状态。在组织损伤，然而，神经细胞可下调EXP这些配体的ression，除去小胶质细胞活化的抑制效果。小胶质细胞的功能（包括吞噬），因此紧密相连的微环境8。然而，到目前为止，还没有标准的测定法来研究小胶质细胞吞噬作用在生理环境或在完全复制其中枢神经系统微环境的一种方式。
几个实验已经开发来测量体外 ，其中primary胶质或小胶质细胞系是与靶细胞（ 例如 ，神经细胞凋亡），或荧光标记的珠培养的小胶质细胞的吞噬活性。那么目标摄取使用荧光显微成像或者流式细胞仪9-12评估。这些检测允许药理或遗传操纵可能会如何影响小胶质细胞的吞噬功能，虽然信息量大，无法完全复制体内环境的复杂的测试。审查小胶质细胞吞噬间接的方法在体内已经报道：这些是由被认为参与在吞噬作用（ 例如，CD68）分子的染色来实现，评估小胶质细胞和目标吞噬（ 例如 ，受损的神经元或突触元素）的物理接近度，或通过吞噬的免疫组化检测小胶质细胞内的目标（ 例如，Aβ）13-17。两项研究使用更直接的方法来评估在体内的小胶质细胞的吞噬功能。休斯和他的同事用成像技术来衡量通过颅内航线18条交付珠小胶质细胞摄取。塞拉等人开发了一种精制方法定量评估使用复杂的成像技术4凋亡细胞的小胶质细胞吞噬作用。然而，这些方法涉及用于组织制备，切片，成像和分析复杂的协议。我们在以前使用的流式细胞仪分析，以评估感光吞噬了通过在培养19视网膜色素上皮（RPE）细胞器段。这里，我们描述了协议来快速评估摄取荧光标记颗粒视网膜小胶质细胞在体内的小胶质细胞的吞噬作用的定量量度。
我们在这里描述的协议允许在三个关键步骤在不到6小时视网膜小胶质细胞吞噬的可靠和定量测量：（1）荧光标记的粒子的玻璃体内递送，（2）收获并制备视网膜组织的，和（3）流流式细胞仪分析。我们开发的方法是，以评估视网膜的小神经胶质细胞吞噬一个健壮的方法，它可以成功地用于测试各种不同的化合物或遗传操作如何改变在生理设置该键小胶质细胞的功能。由于中枢神经系统的一个专门区域，视网膜是研究小神经胶质细胞功能20的方便模式系统。虽然这种方法是在T开发他的眼睛，我们相信它可以为研究小神经胶质细胞吞噬功能全部神经学家有用。
Protocol
Representative Results
Discussion
有在该方法中三个关键步骤：（1）荧光标记的粒子的玻璃体内注射液; （2）收集和准备视网膜组织;及（3）流式细胞仪分析。我们建议研究实行前履行我们在座的方法，玻璃体内注射。白化小鼠（ 例如 BALB / c）和有色溶液（ 例如，荧光标记的颗粒）可用于针的容易可视化和注入溶液。玻璃体内注射是具有挑战性的，如果不正确执行会导致偏见和不同的结果。与注射技术差相关的常?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Salome Murinello is supported by American Diabetes Association grant #1-16-PDF-072. This work was supported by grants to Martin Friedlander from the National Institutes of Health (National Eye Institute EY11254 and EY22025) and the Lowy Medical Research Institute.
Materials
| Stereomicroscope | Nikon | Discontinued | |
| Hamilton syringe, 600 series | Sigma | 26702 | |
| 33 gauge, Small Hub RN NDL, 0.5 in, point style 4 – 12o | Hamilton | 7803-05 | |
| Zymosan A (S. cerevisiae) BioParticles, Alexa Fluor 488 conjugate | ThermoFisher Scientific | Z-23373 | Prepare immediately before injection |
| DPBS | Corning | 21-030-CV | |
| Dumont #5/45 Forceps | Fine Science Tools | 11251-35 | Need two |
| Dumont #5SF Forceps | Fine Science Tools | 11252-00 | |
| Vannas Spring Scissors – 3mm Cutting Edge | Fine Science Tools | 15000-10 | Curved |
| Neural Tissue Dissociation Kit – Postnatal Neurons | Miltenyi Biotec | 130-094-802 | |
| 5 mL Polystyrene Round-bottom Tube | Falcon | 352054 | |
| 96 well U-bottom plate | Falcon | 353077 | |
| Stain Buffer (BSA) | BD Biosciences | 554657 | |
| CD11b-BV650 Antibody | BioLegend | 101259 | |
| Ly6C-APC-Cy7 | BioLegend | 128025 | |
| Ly6G-PE-Cy7 | BioLegend | 127617 | |
| Propidium Iodide | BD Biosciences | 556463 | |
| Purified anti-mouse CD16/32 Antibody | BioLegend | 101301 |
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