Investigating the Biomechanical and Electrophysiological Responses of Astrocytes to Mechanical Trauma

Traumatic brain injury (TBI) is a known risk factor for neurodegeneration, including Alzheimer’s disease (AD). Astrocytes, the brain’s primary homeostatic glial cells, respond dynamically to injury, and alterations in their calcium signaling have been implicated in both TBI and AD pathology. Human induced pluripotent stem cell (hiPSC)-derived astrocytes cultured in 2D monolayers can be mechanically stretched to mimic strain experienced during TBI, providing a controlled in vitro model to study injury response. However, analyzing astrocytic structural and functional responses in this system presents technical challenges due to complex cell morphology and functional dynamics. To address this, the goal of this work was to develop a set of image analysis tools capable of quantifying injury-induced changes in astrocyte structure and function across a range of experimental conditions. A novel calcium activity metric, the Asynchronous Activity Index (AAI), was developed using a CellProfiler-based pipeline. The AAI quantifies fluctuations in total fluorescence over time to assess population-wide calcium activity, providing an alternative when reliable cell-specific segmentation was not feasible. For all other assays, nuclei served as the primary segmentation masks. Analytical pipelines were customized based on the biological interpretation of each signal, with measurements extracted from nuclear regions or defined spatial domains surrounding them. For cell-specific calcium analysis, a custom MATLAB script was used to exclude dim nuclei that had shifted outside the cytoplasmic boundary during the Calbryte time series, due to the motile nature of astrocytes. The nuclear image was captured as a single static frame, while the Calbryte signal was recorded as a time-lapse sequence, leading to spatial drift. The script filtered traces across paired video segments for temporal consistency. These analysis tools successfully identified statistically significant differences in markers across injury conditions. Results mirrored qualitative visual trends and helped reveal whether strain- and time-dependent changes in astrocyte behavior were present and experimentally repeatable. The biologically informed pipelines developed in this work provide a framework for quantifying astrocyte injury responses that will support future studies of astrocyte mechanobiology and calcium signaling in the context of TBI.