Matrix stiffness-dependent microglia activation in response to inflammatory cues: in situ investigation by scanning electrochemical microscopy

Microglia play a crucial role in maintaining the homeostasis of the central nervous system (CNS) by sensing and responding to mechanical and inflammatory cues in their microenvironment. However, the interplay between mechanical and inflammatory cues in regulating microglia activation remains elusive. In this work, we constructed in vitro mechanical-inflammatory coupled microenvironment models of microglia by culturing BV2 cells (a murine microglial cell line) on polyacrylamide gels with tunable stiffness and incorporating a lipopolysaccharide (LPS) to mimic the physiological and pathological microenvironment of microglia in the hippocampus. Through characterization of activation-related proteins, cytokines, and reactive oxygen species (ROS) levels, we observed that the LPS treatment induced microglia on a stiff matrix to exhibit overexpression of NOX2, higher levels of ROS and inflammatory factors compared to those on a soft matrix. Additionally, using scanning electrochemical microscopy (SECM), we performed in situ characterization and discovered that microglia on a stiff matrix promoted extracellular ROS production, leading to a disruption in their redox balance and increased susceptibility to LPS-induced ROS production. Furthermore, the respiratory activity and migration behavior of microglia were closely associated with their activation process, with the stiff matrix-LPS-induced microglia demonstrating the most pronounced changes in respiratory activity and migration ability. This work represents the first in situ and dynamic monitoring of microglia activation state alterations under a mechanical-inflammatory coupled microenvironment using SECM. Our findings shed light on matrix stiffness-dependent activation of microglia in response to an inflammatory microenvironment, providing valuable insights into the mechanisms underlying neuroinflammatory processes in the CNS.