Shear-induced phenotypic transformation of microglia in vitro

Abstract

Brain cells are influenced by continuous fluid shear stress driven by varying hydrostatic and osmotic pressure conditions, depending on the brains pathophysiological conditions. While all brain cells are sensitive to the subtle changes in various physicochemical factors in the microenvironment, microglia, the resident brain immune cells, exhibit the most dramatic morphodynamic transformation. However, little is known about the phenotypic alterations in microglia in response to the changes in fluid shear stress. In this study, we first established a flow-controlled microenvironment to investigate the effects of shear flow on microglial phenotypes, including morphology, motility, and activation states. Microglia exhibited two distinct morphologies with different migratory phenotypes in a static condition: bipolar cells that oscillate along their long axis and unipolar cells that migrate persistently. When exposed to flow, a significant fraction of bipolar cells showed unstable oscillation with an increased amplitude of oscillation and a decreased frequency, which consequently led to the phenotypic transformation of oscillating cells into migrating cells. Interestingly, the level of pro-inflammatory genes increased in response to shear stress, while there were no significant changes in the level of anti-inflammatory genes. Our findings suggest that an interstitial fluid-level stimulus can cause a dramatic phenotypic shift in microglia toward pro-inflammatory states, shedding light on pathological outbreaks of severe brain diseases. Given that the fluidic environment in the brain can be locally disrupted in pathological circumstances, the mechanical stimulus by a fluid flow should also be considered a crucial element in regulating the immune activities of the microglia in brain diseases. Statement of SignificanceCellular morphology and motility are important factors that encompass the alterations in protein and gene-level expressions within cells. In pathological conditions, microglia, the resident brain immune cells, are known to undergo morphodynamic transformations in response to various physicochemical stimuli. Besides the commonly known soluble biochemical factors in the microenvironment, the differential flow characteristics of ISF have been linked to several neurological diseases, such as Alzheimers, Parkinsons, and brain tumors. Microglial cells, which are extremely sensitive to subtle changes in extracellular stimuli, have been identified as key players in these pathological conditions. Despite its importance, however, it has been challenging to study the sole effect of a shear flow on microglia. We investigated the morphodynamic features of microglia in response to precisely controlled interstitial-level fluid flow conditions using a microfluidic system in which isolated microglia are monitored in real-time while the undesirable effects from other extracellular factors are minimized.