Imaging and Manipulating Astrocyte Function In Vivo in the Context of CNS Injury
Imaging and Manipulating Astrocyte Function In Vivo in the Context of CNS Injury
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DOI：
10.1007/978-1-4939-9068-9_16
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发表时间：
2019-01-01
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影响因子：
--
通讯作者：
Robel, Stefanie
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文献类型：
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作者：
Shandra, Oleksii;Robel, Stefanie
星形胶质细胞是在健康和患病的中枢神经系统（CNS）中执行复杂的稳态功能的胶质细胞。到目前为止，还不可能可靠地培养成体星形胶质细胞，并且在正常环境之外对星形胶质细胞的研究结果难以解释。因此，大多数培养研究使用从出生后啮齿动物分离的星形胶质细胞。然而，培养的星形胶质细胞不显示其复杂的三维体内形态，并且培养的星形胶质细胞的转录组与急性分离的星形胶质细胞的转录组显著不同（Cahoy等人，J Neurosci 28：264-278，2008）。用于培养实验的星形胶质细胞分离，以及急性脑切片的切割，诱导星形胶质细胞类似于严重急性损伤的反应性。在对CNS损伤（例如中度或重度局灶性创伤性脑损伤（TBI））的响应中，星形胶质细胞可以在称为反应性星形胶质细胞增生的过程中改变细胞数量、生理状态、基因和蛋白质表达、分泌组和形态。这使得使用固有地诱导星形胶质细胞增生的方法（例如，分离脑组织用于培养或脑切片用于急性脑切片）具有挑战性，特别是当研究条件存在星形胶质细胞功能变化时，星形胶质细胞功能变化更温和和/或性质不同。在本方法章节中，我们将描述一种技术方法，该方法允许使用双光子体内成像研究完整脑中的星形胶质细胞。我们将使用轻度TBI作为如何使用该方法比较损伤前后同一动物中星形胶质细胞功能的示例。J Biomed Opt 16：075003，2011）使用光学飞秒脉冲激光激活来增加星形胶质细胞Ca 2+。我们将提供手术技术的系统指导，如果操作得当，可以在损伤前以及损伤后的几天，几周甚至几个月内对同一实验动物进行体内星形胶质细胞成像。我们还将详细阐述星形胶质细胞Ca 2+成像的挑战，以及不同的图像采集设置如何影响星形胶质细胞Ca 2+振荡的读数。
Astrocytes are glial cells carrying out complex homeostatic functions in the healthy and diseased central nervous system (CNS). It has so far been impossible to reliably culture adult astrocytes and the results of studies on astrocytes outside of their normal environment are challenging to interpret. Consequently, most culture studies use astrocytes isolated from postnatal rodents. Yet cultured astrocytes do not display their complex three-dimensional in vivo morphology, and transcriptomes of cultured astrocytes vary significantly from those of acutely isolated astrocytes (Cahoy et al., J Neurosci 28:264-278, 2008). Astrocyte isolation for culture experiments, and the cutting of acute brain slices, induces astrocyte reactivity similar to a severe acute injury. In response to CNS injury, such as moderate or severe focal traumatic brain injury (TBI), astrocytes can change in cell number, physiological state, gene and protein expression, secretome, and morphology, in a process termed reactive astrogliosis. This makes the use of methods that inherently induce astrogliosis (e.g., dissociation of brain tissue for culture or sectioning of brains for acute brain slices) challenging, especially when conditions are studied that present with changes in astrocyte function that are milder and/or of a different nature.In this methods chapter, we will describe a technical approach that allows one to study astrocytes in the intact brain using two-photon in vivo imaging. We will use mild TBI as an example of how to use this approach to compare astrocyte function in the same animal before and after an injury.Here we describe the use of a noninvasive label-free method (Choi et al., J Biomed Opt 16:075003, 2011) to increase astrocyte Ca2+ using optical femtosecond pulsed laser activation. We will provide systematic instruction of the surgical technique, which when done properly, allows in vivo astrocyte imaging in the same experimental animal before the injury as well as over the course of days, weeks, and even months after injury. We will also elaborate on challenges in astrocytic Ca2+ imaging and how different image acquisition settings can affect the readout of astrocyte Ca2+ oscillations.