Imaging and Manipulating Astrocyte Function In Vivo in the Context of CNS Injury
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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.
Key wordsAstrocytes Astrogliosis In vivo imaging Multiphoton Two-photon Microscopy Traumatic brain injury Calcium
This work was supported by the National Institute of Neurological Disorders and Stroke at the National Institutes of Health (grant number R01NS105807).
3D reconstruction of astrocytes and TRITC-Dextran labeled vasculature before TBI. Imaging laser wavelength 870 nm. Zoom factor 1.5 (MP4 4684 kb)
3D reconstruction of astrocytes and TRITC-Dextran labeled vasculature 1 day after TBI. Imaging laser wavelength 870 nm. Zoom factor 1.5 (MP4 4583 kb)
3D reconstruction of astrocytes and TRITC-Dextran labeled vasculature 3 days after TBI. Imaging laser wavelength 870 nm. Zoom factor 1.5 (MP4 4642 kb)
Astrocyte endfeet laser stimulation cause Ca2+ wave generation and constriction of the arteriole. Imaging wavelength 870 nm 10% laser power. Stimulation laser power 30%, duration 100 ms. Zoom factor 1.3. Red line indicates the laser stimulation ROI (MP4 6903 kb)
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