Cerebral Blood Flow and Metabolism (Japanese journal of cerebral blood flow and metabolism)
Online ISSN : 2188-7519
Print ISSN : 0915-9401
ISSN-L : 0915-9401
Original Articles
Long-term repeated in vivo real-time analysis of microglia in the mouse cortex with a closed-cranial window by Tomita-Seylaz method
Hiroya YukiKazuto MasamotoMiyuki UnekawaYutaka TomitaIwao KannoNorihiro Suzuki
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2017 Volume 28 Issue 2 Pages 249-256

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Abstract

Microglia, known as the primary immune cell of central nervous system (CNS), plays a crucial role in maintaining homeostasis of the brain tissue by dynamically surveying their cellular environment during normal development, function, and repair from the injury. In the present study, we aimed to establish a method for long-term repeated real-time imaging and analysis of microglia in the mouse cortex in vivo. To this end, genetically-modified mice (CX3CR1-GFP mice, the Jackson Laboratory, 17–33 g, N=13) in which the cortical microglia expresses green fluorescence protein (GFP) were used for the experiments. A part of the skull was replaced with a closed-cranial window by using a Tomita-Seylaz method for transparency. The GFP-positive microglia and cortical microvessels labeled with sulforhodamine 101 (SR101) were imaged with two-photon laser scanning fluorescence microscopy repeatedly up to 3 weeks after the cranial surgery. For quantification of the cellular structure and motility, cell size, number density, and number of fine processes of the GFP-positive microglia were measured with custom-written software. We observed that consistent morphology and clear visibility of the GFP-positive microglia was maintained up to a depth of 800 μm from the cortical surface during entire periods of the experiment. There were no detectable differences in the soma area (32±2 μm2), number density (6–8×103 number/mm3), and number of processes (10.3±0.5 number/cell) of the GFP-positive microglia between 1–3 weeks following installation of the cranial window. In conclusion, the present methods provide stable and reproducible real-time measures of the morphology and motility of microglia in vivo in the living mouse cortex. The present results are beneficial for use as a control data to further understand the role and behavior of microglia in neurodegenerative diseases, such as ischemia, chronic hypoperfusion, and dementia.

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© 2017 The Japanese Society of Cerebral Blood Flow and Metabolism
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