Abstract
Two-photon imaging of fluorescently labeled microglia in vivo provides a direct approach to measure motility of microglial processes as a readout of microglial function that is crucial in the context of neurodegenerative diseases, as well as to understand the neuroinflammatory response to implanted substrates and brain-computer interfaces. In this longitudinal study, we quantified surveilling and photodamage-directed microglial processes motility in both acute and chronic cranial window preparations and compared the motility under isoflurane and ketamine anesthesia to an awake condition in the same animal. The isoflurane anesthesia increased the length of surveilling microglial processes in both acute and chronic preparations, while ketamine increased the number of microglial branches in acute preparation only. In chronic (but not acute) preparation, the extension of microglial processes toward the laser-ablated microglial cell was faster under isoflurane (but not ketamine) anesthesia than in awake mice, indicating distinct effects of anesthetics and of preparation type. These data reveal potentiating effects of isoflurane on microglial response to damage, and provide a framework for comparison and optimal selection of experimental conditions for quantitative analysis of microglial function using two-photon microscopy in vivo.
Highlights
Microglia are highly dynamic immune cells of the central nervous system which have a wide range of functions and play important roles in brain physiology and pathologies
The main findings of this study are the significant effects of both the preparation type and the anesthetics on microglial morphology, motility, and response to damage
The potentiating effect of isoflurane on microglial motility that we observed using in vivo imaging is in sharp constant with the motility-suppressing effect of isoflurane observed previously using ex vivo and in vitro imaging (Madry et al, 2018), once again highlighting the need for caution when extrapolating the results of ex vivo/in vitro experiments to in vivo conditions
Summary
Microglia are highly dynamic immune cells of the central nervous system which have a wide range of functions and play important roles in brain physiology and pathologies. Two-Photon Imaging of Microglia occurring small lesions, ranging from ruptured micro-capillaries to dying cells releasing toxic content into tightly controlled extracellular space (Kraft and Harry, 2011; Nayak et al, 2014). This protective function relies on at least two distinct types of motility of microglial processes: (i) surveilling motility, and (ii) damage-directed motility. The surveilling, a.k.a. resting, motility refers to surveillance of the microglial cell’s immediate environment, which enables timely detection of brain damage and pathogen infiltration (Hanisch and Kettenmann, 2007) This surveilling motility crucially depends on the membrane potential and requires the constitutive hyperpolarizing activity of a specific type of inwardly-rectifying potassium channel called TWIK-related Halothane-Inhibited K+ channel, THIK-1 (Madry et al, 2018). Microglial surveillance can be selective/directional in the sense that microglial cells monitor and interact with neurons during conditions of cerebral calcium reduction in the normal and diseased brain (Eyo et al, 2015)
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