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视网膜胶质细胞活化的体内动力学在神经退行性疾病：共聚焦成像眼底及细胞形态计量小鼠青光眼

Published: May 11, 2015
doi:
10.3791/52731
, , ,
1Department of Neurobiology & Anatomy,University of Utah, 2Department of Ophthalmology & Visual Sciences,University of Utah

Summary

小胶质细胞活化和小神经胶质细胞是关键应对慢性神经退行性病变。这里，我们提出在体内 ，视网膜CX3CR1-GFP +胶质细胞共聚焦检眼镜的长期可视化方法，以及用于阈和形态分析，以确定和量化它们的活化。我们监测期间与年龄有关的青光眼的早期阶段小胶质细胞的变化。

Abstract

小胶质细胞，这是CNS驻地神经免疫细胞，改变其形态和大小响应中枢神经系统损伤，切换到激活状态具有不同的功能和基因表达谱。在健康，损伤和疾病胶质细胞活化的作用仍然不完全是由于他们的动态和复杂的调节反应的变化在他们的微环境的理解。因此，它是对非侵入性地监控关键和分析在完整生物体随时间的变化小胶质细胞活化。在体内胶质细胞活化的研究已经被推迟了技术限制，以跟踪小胶质细胞的行为没有改变中枢神经系统环境。这已经在慢性神经变性，在那里长期变化必须跟踪是特别具有挑战性的。视网膜，中枢神经系统器官适合于非侵入性的实时成像，提供了强大的系统，以可视化和在慢性病症表征小胶质细胞活化的动力学。

<p cl屁股="“jove_content”">此协议概括了长期的， 在体内成像的视网膜小胶质细胞，使用共聚焦检眼镜（CSLO）和CX3CR1 GFP / +报告小鼠，以可视化的小胶质细胞与细胞的分辨率的方法。此外，我们描述的方法来量化细胞活化和密度大细胞亚群每月改变（每视网膜200-300个细胞）。我们确认使用常染色体区域的作为用于在视网膜小胶质细胞活化的活跟踪一个有用的度量通过施加在体内图像的自动阈值基于形态测定分析。我们使用这些实时图像采集和分析的策略中的视网膜神经变性的慢性青光眼的小鼠模型的早期阶段，以监测小胶质细胞活化和小神经胶质细胞的动态变化。这种方法应该是有用，调查小胶质细胞在慢性中枢神经系统疾病，影响视网膜和视神经的神经元和轴突下降的贡献。

Introduction

小胶质细胞是自早期胚胎发育和整个成年期，专门驻留在中枢神经系统（CNS）神经免疫细胞。配备有受体的复杂剧目，小胶质细胞的活性和区域的异质性是由他们的双向相互作用调节与相邻的神经元，神经胶质细胞，血-脑屏障和渗透神经炎性细胞1,2-。小胶质细胞基础功能有助于生理保养和维修，因为他们品尝其领土的动态平衡3,4扰动。在中枢神经系统损伤或疾病，小胶质细胞是第一个响应者的神经信号，然后触发他们过渡到无功表型，被称为“激活小神经胶质细胞2,5-7。小胶质细胞活化涉及基因和蛋白的表达，其耦合到细胞胞体和过程调整大小和重塑6-9的一个复杂的循环。小胶质细胞的活化，以及细胞和再分配聚类，可伴随在细胞的数量（称为小神经胶质细胞）的本地整体增加。这可能是由于细胞增殖和自我更新，有或没有招募的血液衍生的单核细胞3,4,7,10-14。在范围广泛的年龄依赖性，慢性CNS疾病，持续小神经胶质细胞和小胶质细胞活化平行疾病进展15-19。小胶质细胞是如何影响神经退行性疾病仍不清楚，主要是因为他们玩，可能有疾病发生，发展多样化的贡献都神经保护和有害的作用。实时成像旨在了解的慢性CNS疾病监测了动物模型和人类的受损中枢神经系统的小神经胶质的行为，并证明了小胶质细胞的改变是可检测的开始在早期疾病阶段15-17,19,20。因此，关键是要开发的方法来检测和监测体内小胶质细胞活化。

无创DETE在脑小胶质细胞激活区域变化ction确立为在神经变性疾病的进展体内指示器的重要，使用分子成像或生物发光和正电子发射断层扫描或磁共振成像18,21,22。这些高度量化和非侵入性的分子和核成像方法检测胶质细胞增生与区域分辨率。或者，在CX3CR1 GFP / +小鼠的双光子共焦成像已经允许大脑胶质细胞的观察与细胞分辨率3,4,9,20,23-28。然而，这种方法限制了长期和反复观察慢性小胶质改建，由下式给出甚至微创脑成像程序29干扰它们的行为的潜在风险。或者，视网膜提供了直接的最佳条件， 在体内的可视化和小胶质细胞在其完整的CNS利基整个老化重复监控，急性损伤，并潜在期间慢性神经变性疾病。因此，最近的研究证明了视网膜小胶质细胞表达GFP的高分辨率成像的可行性，通过调整共焦激光扫描眼底镜（CSLO）图像实时CX3CR1 GFP / +小鼠。这已被用于跟踪在个体小鼠长达10周的GFP +细胞数每周改变以下急性诱发损伤或高眼压30-36。

我们已经扩展这种方法来进行长期的成像历时数月，并定量跟踪的基础上利用形态分析胞体大小小胶质细胞活化的变化。常染色体的大小被定义为小胶质细胞活化在实时成像研究使用皮质切片双光子激光共聚焦显微镜 CX3CR1-GFP +小胶质9 体内成像执行有用的指标。这些和其他的研究也证实常染色体大小和Iba1蛋白表达水平之间的相关性，的wh脑出血也随着活化9,37。因此，活化的小胶质细胞可在活体小鼠来鉴定，并且它们的数量和分配过程中的CNS健康和疾病随时间监测。

这个协议描述为CSLO实时图像采集和分析方法，监测小胶质细胞的数量，视网膜神经节细胞（RGC）变性（ 图1）中的分布和形态的活化。因此，本研究的用途：1）遗传性青光眼（DBA / 2J），该发生年龄依赖性视神经和视网膜神经变性和小鼠模型显示5和10个月38,39年龄之间显着的可变性疾病进展，2）每月CSLO 体内成像的GFP +细胞在视网膜长期可视化和杂CX3CR1 GFP / + DBA / 2J小鼠的年龄3-5个月，3）由分段和阈值实时成像分析以分离细胞胞体和测量无髓鞘视神经该IR区。施加这些策略在慢性青光眼的早期阶段，以评估视网膜小胶质细胞激活状态的动力学。

Protocol

在活体成像使用犹他州大学批准的机构动物护理和使用委员会协议无致病菌设施进行。 注意：此成像协议用于报告小鼠中视网膜小胶质细胞和浸润的单核细胞/巨噬细胞表达的fractalkine的受体基因座（CX3CR1）的控制下，绿色荧光蛋白（GFP）。 1. 在视网膜GFP +小胶质细胞的体内成像共聚焦扫描激光眼底镜检查（CSLO） 转水控制的加热系统，建立稳…

Representative Results

我们最近在体内的研究中使用这些实时图像采集和分析方法，以可视化，并在慢性青光眼和他们的关系的早期阶段跟踪动力学和视盘和视网膜小胶质细胞的变化模式来晚了神经退行性疾病59。在这里，我们示出了CSLO图像采集协议跨越大面积在个别年轻杂CX3CR1-GFP的DBA / 2J视网膜中央视网膜（ 图5）的可视化的小神经胶质细胞。基于高元分辨率，我们应用实时图像阈值和形?…

Discussion

神经变性疾病中在线监测的小胶质细胞数目和形态的激活需要使用的非侵入性的成像方法，使细胞的功能的详细可视化。成像后，小胶质细胞，必须分离（分段），用于形态测定分析通过使用多个阈值的步骤，以评估常染色体的大小和/或过程的复杂性作为读数为小胶质细胞活化。在这个协议中，我们描述了使用CSLO实时图像采集，和小胶质细胞活化的基于序列的细胞和胞体分割的定量分析方法。?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank the Scientific Computing and Imaging Institute of the University of Utah for use of FluoRender software (R01GM09815). This work was supported by grants from the Glaucoma Research Foundation, Melsa M. and Frank Theodore Barr Foundation and the US National Institute of Health, (R01EY020878 and R01EY023621) to M.L.V., and (R01EY017182 and R01EY017950) to B.K.A.

Materials

Name Company Catalog Number Comments
DBA/2J and
CX3CR1-GFP/+ mice		 The Jackson Laboratory, Bar Harbor, ME 000671 and 00582 Mice are breed in-house, introducing new breeders every 3 to 4 generations to prevent genetic drift.
301/2G needle and 1 ml tuberculin slip-tip syringe BD, East Rutherford, NJ 305106 and 309659
2,2,2-tribromoethanol, tert-Amyl alcohol 99% and phosphate buffer saline tablets Sigma-Aldrich, St. Louis, MO T48402, 152463 and P4417 Avertin solution (pH 7.3, sterile filtered) must be freshly made solution or stored at 4ºC for up to 1 week.
Heat therapy T/Pump Gaymar Industries, Orchard Park, NY TP-650
Cotton-tipped applicators Fisher Scientific, Pittsburgh, PA 23-400-100
Tropicamide 1%  Bausch & Lomb, Rochester, NY NDC 24208-585-64
PMMA contact lenses, 1.70-base curve, 3.2 mm total diameter, 0.40 mm-thick center Cantor & Nissel Ltd., Northamptonshire, UK G003709 Lenses are rinsed with sterile PBS and stored in polypropylene boxes.
HRA/Spectralis confocal scanning laser ophthalmoscope and Eye Explorer software Heidelberg Engineering GmbH, Carlsbad, CA Version 1.7.1.0
PowerPoint Microsoft, Redmond, WA Version 14.4.3
Adobe Photoshop Adobe, San Jose, CA Version CS3
FluoRender Scientific Computing and Imaging Institute, University of Utah Version 2.13 Freeware. http://www.sci.utah.edu/software/13-software/127-fluorender.html
NIS-Elements C Nikon, Melville, NY Version 4.30.01
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Tags

Keywords: Retinal MicrogliaIn Vivo ImagingConfocal OphthalmoscopyCX3CR1GFP/+ Reporter MiceMicroglial ActivationMicroglial MorphometryChronic NeurodegenerationGlaucomaRetinal NeurodegenerationOptic Nerve
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Bosco, A., Romero, C. O., Ambati, B. K., Vetter, M. L. In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma. J. Vis. Exp. (99), e52731, doi:10.3791/52731 (2015).
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