Shandra Oleksii, Robel Stefanie
Virginia Tech Carilion Research Institute, Roanoke, VA, USA.
School of Neurosciences, Virginia Tech, Blacksburg, VA, USA.
Methods Mol Biol. 2019;1938:233-246. doi: 10.1007/978-1-4939-9068-9_16.
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 Ca 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 Ca imaging and how different image acquisition settings can affect the readout of astrocyte Ca oscillations.
星形胶质细胞是在健康和患病的中枢神经系统（CNS）中执行复杂稳态功能的神经胶质细胞。到目前为止，可靠地培养成年星形胶质细胞是不可能的，并且在其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新生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会呈现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有显著差异（Cahoy等人，《神经科学杂志》28:264 - 278，2008）。用于培养实验的星形胶质细胞分离以及急性脑片的切割会诱导星形胶质细胞反应，类似于严重的急性损伤。响应中枢神经系统损伤，如中度或重度局灶性创伤性脑损伤（TBI），星形胶质细胞在细胞数量、生理状态、基因和蛋白质表达、分泌组以及形态方面会发生变化，这个过程称为反应性星形胶质细胞增生。这使得使用固有诱导星形胶质细胞增生的方法（例如，为培养而解离脑组织或为急性脑片而切片脑组织）具有挑战性，特别是当研究的条件呈现出星形胶质细胞功能的变化较为轻微和/或性质不同时。在本章方法中，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在完整大脑中研究星形胶质细胞。我们将以轻度TBI为例，说明如何使用这种方法比较同一动物在损伤前后的星形胶质细胞功能。在这里，我们描述使用一种非侵入性的无标记方法（Choi等人，《生物医学光学杂志》16:075003，2011）通过光学飞秒脉冲激光激活来增加星形胶质细胞的钙。我们将提供手术技术的系统指导，操作得当的话，该技术允许在损伤前以及损伤后的数天、数周甚至数月内在同一实验动物中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钙成像中的挑战以及不同的图像采集设置如何影响星形胶质细胞钙振荡的读数。
