Methods for Analyzing Microglial Signaling and Cortical Spreading Depression Wavefronts in 2-Photon Calcium Images

Cortical spreading depressions are a neurological phenomenon that occur most notably during strokes and traumatic brain injury, characterized by slowly propagating waves of near-complete neuron depolarization followed by prolonged suppression of neuron activity. While their cause and purpose is still poorly understood, it is believed that they play a role in activating microglia, leading to the release of pro-inflammatory cytokines into the brain. Microglial activation is also believed to promote the occurrence of further cortical spreading depressions. The added neurological stress and inflammation induced by cortical spreading depressions can exacerbate injury to the brain. As a result, suppressing their generation and downstream effects has become a topic of interest in medical research, with microglia as a potential target. In this effort, 2-photon fluorescence microscopy and calcium imaging has been used in recent years to generate image sequences of cortical spreading depression propagation and subsequent microglial activation through calcium-based signaling. Due to the novelty of such images, methods for consistently analyzing such image sequences have not been presented within the literature. This thesis contributes to solving this issue in two distinct ways. The first is by presenting a workflow for segmenting microglia and measuring their calcium-based signaling in response to cortical spreading depressions. The second is a novel image segmentation algorithm for segmenting the wavefront boundary of cortical spreading depressions.