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Characterising microglial activation: refinement and validation of primary in vitro models

Al-Shaheeb, Sama

Characterising microglial activation: refinement and validation of primary in vitro models Thumbnail


Authors

Sama Al-Shaheeb



Abstract

Background: Neuroinflammation is common to many neurodegenerative conditions and is principally mediated by microglia. Microglia maintain homeostasis, respond to trauma/infection and can promote repair. This is achieved by switching between surveillance (M0), pro-inflammatory (M1) and pro-repair (M2) phenotypes. However, inappropriate phenotype switching contributes to neurodegeneration, through failure to clear harmful substances or even direct tissue damage. How inappropriate switching occurs is not fully understood. Therefore, identifying a model for assessing phenotypic changes would provide a powerful tool to investigate the roles of microglia in disease, and develop therapies based on microglial immunomodulation. Nanoparticles (NPs) may be key to delivering immunomodulatory drugs to the brain, but it is unknown whether NPs will provoke inflammatory microglial responses.

Methods: RT-qPCR was used to detect differences in gene expression for primary rat microglia, in response to various culture conditions and stimuli, including differently formulated NPs (e.g., PEGylated ‘stealth’ NPs and dextran ‘control’ NPs).

Results: Three microglia reference genes were validated for qPCR (Gapdh, Usp14 and Rpl32). The presence of serum did not alter microglial activation state, compared to two commercially-available serum-free media. Cardinal M1 markers (Il-6, Il-1ß and Tnf-a) were well-characterised in response to a wide range of lipopolysaccharide (LPS) concentrations, and at three different time points (2, 6 and 24 h). Unexpectedly, several pro-M2 stimuli (Il-4, Il-13 and Tgf-ß) failed to alter M2 markers, even with multiple changes to the protocols. Microglia exhibited uptake of several NP types without inflammatory responses.

Conclusions: An in vitro model of microglial switching has been refined and validated for M1 responses. Further refinement is necessary for study of M2 switching. The NP data suggest that NPs could be used without unintended inflammatory responses.

Publicly Available Date Mar 28, 2024

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