Abstract
PURPOSE: Microglia, the brain's immune cells, play important roles in neuronal differentiation, survival, and death. The function of microglia is deeply related to the morphologies; however, it is too complex to observe conventionally and identify the condition of living microglia using optical microscopes. Herein, we proposed a new method to observe living cultured microglia and their reactivity to inflammation via the acoustic impedance mode of a scanning acoustic microscope. METHODS: Primary cultured microglia collected from rat pups exposed to acetamiprid, an insecticide, in utero were observed with both acoustic interface impedance mode (C-mode) and transparent three-dimensional impedance mode (B-mode). RESULTS: We characterized microglia into four types based on the results obtained from acoustic impedance, cytoskeletal information, and laser confocal imaging. Biphasic acoustic observation using B-mode and C-mode gave us information regarding the dynamic morphologies of living microglia treated with adenosine triphosphate (ATP) (600 μmol/L), which reflects distress signals from inflamed neurons. Acetamiprid exposure induced microglia response even in the neonatal period. ATP stimulus altered the shape and thickness of microglia with a change in the bulk modulus of the cell. Three-dimensional alteration with ATP stimulus could be observed only after biphasic acoustic observation using B-mode and C-mode. This acoustic observation was consistent with confocal observation using anti-Iba-1 and P2Y12 immunocytochemistry. CONCLUSION: This study demonstrated the adequacy of using a scanning acoustic microscope in analyzing microglia's shape, motility, and response to inflammation.