35: Quantifying Extracellular Matrix?Mediated BV2 Cell Morphological Changes in Pain Models

Topic: Basic Science
Subtopic: Non-specific to any diagnosis (including studies in healthy animals), Developing methodology/technology, Neuroimmunology

Background and Aims: The extracellular matrix (ECM) provides mechanical cues that shape how cells spread, migrate, and respond to injury. Microglia, key mediators of neuroinflammation and central sensitization, dynamically remodel the ECM and influence synaptic plasticity in pain pathways. Tissue stiffness changes in conditions such as neuropathic pain, tumors, and scarring, but the quantitative effects of matrix rigidity and inflammation on microglial morphology—as a proxy for state and function—are unclear. In this study, we developed and applied a scalable CellProfiler-based workflow to define how ECM stiffness and lipopolysaccharide (LPS) stimulation alter BV2 microglial morphology, providing an efficient and objective workflow for morphological analysis.

Methods: BV2 microglia were cultured on collagen and polydimethylsiloxane substrates spanning physiologically relevant stiffnesses (0.2–32 kPa). Cells were treated with LPS to model pain-related neuroinflammation or left untreated, then fixed after 24 hours. Phase-contrast images (Keyence BZ-X810, 4x/0.20 objective) were analyzed using a custom CellProfiler pipeline that computationally segmented somas and quantified area, circularity, and a Hu-moment–derived symmetry metric. Two-way ANOVA with Tukey’s post hoc tests assessed effects of ECM stiffness and LPS stimulation on each measure, examining how inflammatory and mechanical cues relevant to chronic pain shape microglial morphology.

Results: Microglial somal area demonstrated a stiffness-dependent parabolic relationship, peaking at 8 kPa, with LPS increasing area across all ECM rigidities and attenuating this peak. Symmetry followed a similar trend, peaking at 16 kPa without LPS and 2 kPa with LPS. In both conditions, symmetry was lowest at 32 kPa. LPS stimulation reduced overall symmetry and shifted peak symmetry toward healthy ECM stiffness levels. Circularity also varied parabolically with ECM stiffness, peaking near healthy brain values at 2 kPa under LPS, whereas unstimulated cells exhibited a weak relationship between ECM stiffness and circularity.

Conclusions: The ECM and inflammation jointly shape microglial morphology in a dynamic and nonlinear manner. LPS stimulation amplifies microglial sensitivity to matrix rigidity, promoting larger and less symmetric morphologies at softer stiffnesses resembling healthy or early-injury brain tissue. Such morphologies can potentially enhance or disrupt immunosurveillance and synaptic modulation in pain pathways. These findings support a mechanobiological link between tissue stiffness, microglial state, and pain-related plasticity, highlighting the need for further focus on extra-neuronal factors such as glia and the ECM in pain research. Additionally, this research supports the practicality of efficient and quantitative-based workflows for morphology analysis in pain research.

References: Crapser, J.D., Arreola, M.A., Tsourmas, K.I. et al. Microglia as hackers of the matrix: sculpting synapses and the extracellular space. Cell Mol Immunol 18, 2472–2488 (2021). https://doi.org/10.1038/s41423-021-00751-3

Soles, A., Selimovic, A., Sbrocco, K., Ghannoum, F., Hamel, K., Moncada, E. L., Gilliat, S., & Cvetanovic, M. (2023). Extracellular Matrix Regulation in Physiology and in Brain Disease. International Journal of Molecular Sciences, 24(8), 7049. https://doi.org/10.3390/ijms24087049

Stirling, D. R., Swain-Bowden, M. J., Lucas, A. M., Carpenter, A. E., Cimini, B. A., & Goodman, A. (2021). CellProfiler 4: improvements in speed, utility and usability. BMC Bioinformatics, 22(1). https://doi.org/10.1186/s12859-021-04344-9

Presenting Author: Giuliano Vitale
Submitting Author: Giuliano Vitale

Senior Author: Vaneeza Kausar

IASP 2026 Global Year theme of Neuropathic Pain: No