Abstract
Traumatic brain injury (TBI) results in a cascade of cellular responses, which produce neuroinflammation, partly due to microglial activation. Transforming from surveying to primed phenotypes, microglia undergo considerable molecular changes. However, specific microglial profiles in rat remain elusive due to tedious methodology and limited availability of reagents. Here, we present a flow cytometry-based analysis of rat microglia 24 h after TBI using the controlled cortical impact model, validated with a bioinformatics approach. Isolated microglia are analyzed for morphological changes and their expression of activation markers using flow cytometry, traditional gating-based analysis methods and support the data by employing bioinformatics statistical tools. We use CD45, CD11b/c, and p2y12 receptor to identify microglia and evaluate their activation state using CD32, CD86, RT1B, CD200R, and CD163. The results from logic-gated flow cytometry analysis was validated with bioinformatics-based analysis and machine learning algorithms to detect quantitative changes in morphology and marker expression in microglia due to activation following TBI.
Highlights
Traumatic brain injury (TBI) results in a cascade of cellular responses, which produce neuroinflammation, partly due to microglial activation
The main population of cells was identified through Forward Scattered light (FSC) and Side Scattered light (SSC)
FSC is a measurement for the relative size of the event, while SSC is a measurement for the event g ranularity[27]
Summary
Traumatic brain injury (TBI) results in a cascade of cellular responses, which produce neuroinflammation, partly due to microglial activation. The results from logic-gated flow cytometry analysis was validated with bioinformatics-based analysis and machine learning algorithms to detect quantitative changes in morphology and marker expression in microglia due to activation following TBI. Activated microglia undergo dramatic morphological transformation[7], which is required for their revised role in the neuroinflammatory response Their newly acquired amoeboid shape makes them indistinguishable from the recruited blood derived macrophages responding damaged tissue[12]. The first subdivision of M2 is when microglia are stimulated using IL4 or IL13 into an alternative pathway that includes immunity against parasites, Th2 cell recruitment, and tissue repair. These conditions define the cells as M2a-polarized. In M2c, cells will overexpress transforming growth factor beta (TGF-β), sphingosine kinase and CD163, the membrane-bound scavenger receptor for haptoglobin/hemoglobin
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