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Neuroinflammation is characterized by the elevation of cytokines and adenosine triphosphate (ATP), which in turn activates microglia. These immunoregulatory molecules typically form gradients in vivo, which significantly influence microglial behaviors such as increasing calcium signaling, migration, phagocytosis, and cytokine secretion. Quantifying microglial calcium signaling in the context of inflammation holds the potential for developing precise therapeutic strategies for neurological diseases. However, the current calcium imaging systems are technically challenging to operate, necessitate large volumes of expensive reagents and cells, and model immunoregulatory molecules as uniform concentrations, failing to accurately replicate the in vivo microenvironment. In this study, we introduce a novel calcium monitoring micro-total analysis system (CAM-μTAS) designed to quantify calcium dynamics in microglia (BV2 cells) within defined cytokine gradients. Leveraging programmable pneumatically actuated lifting gate microvalve arrays and a Quake valve, CAM-μTAS delivers cytokine gradients to microglia, mimicking neuroinflammation. Our device automates sample handling and cell culture, enabling rapid media changes in just 1.5 s, thus streamlining the experimental workflow. By analyzing BV2 calcium transient latency to peak, we demonstrate location-dependent microglial activation patterns based on cytokine and ATP gradients, offering insights contrasting those of non-gradient-based perfusion systems. By harnessing advancements in microsystem technology to quantify calcium dynamics, we can construct simplified human models of neurological disorders, unravel the intricate mechanisms of cell-cell signaling, and conduct robust evaluations of novel therapeutics. 

The highly controlled migration of neutrophils toward the site of an infection can be altered when they are trained with lipopolysaccharides (LPS), with high dose LPS enhancing neutrophil migratory pattern toward the bacterial derived source signal and super-low dose LPS inducing either migration toward an intermediary signal or dysregulation and oscillatory movement. Empirical studies that use microfluidic chemotaxis-chip devices with two opposing chemoattractants showed differential neutrophil migration after challenge with different LPS doses. The epigenetic alterations responsible for changes in neutrophil migratory behavior are unknown. We developed two mathematical models that evaluate the mechanistic interactions responsible for neutrophil migratory decision-making when exposed to competing chemoattractants and challenged with LPS. The first model, which considers the interactions between the receptor densities of two competing chemoattractants, their kinases, and LPS, displayed bistability between high and low ratios of primary to intermediary chemoattractant receptor densities. In particular, at equilibrium, we observe equal receptor densities for low LPS (< 15ng/mL); and dominance of receptors for the primary chemoattractant for high LPS (> 15ng/mL). The second model, which included additional interactions with an extracellular signal-regulated kinase in both phosphorylated and non-phosphorylated forms, has an additional dynamic outcome, oscillatory dynamics for both receptors, as seen in the data. In particular, it found equal receptor densities in the absence of oscillation for super-low and high LPS challenge (< 0.4 and 1.1 <LPS< 375 ng/mL); equal receptor densities with oscillatory receptor dynamics for super-low LPS (0.5 < LPS< 1.1ng/mL); and dominance of receptors for the primary chemoattractant for super-high LPS (>376 ng/mL). Predicting the mechanisms and the type of external LPS challenge responsible for neutrophils migration toward pro-inflammatory chemoattractants, migration toward pro-tolerant chemoattractants, or oscillatory movement is necessary knowledge in designing interventions against immune diseases, such as sepsis.