CSF-1 receptor inhibition as a highly effective tool for depletion of microglia in mixed glial cultures

A breakthrough in the microglia and macrophages field was the identification of the macrophage colony stimulating factor-1 (CSF-1) as a pro-survival factor. Its pharmacological inhibition in animals depletes rapidly all microglia and macrophages. Microglial depletion in mixed glial cultures has always represented a challenge and none of the existing approaches delivers satisfactory results.

We applied a CSF-1R inhibitor (PLX5622) in primary mouse glial cultures, analyzing microglial dose-responses, starting at different time-points and incubating for various periods of time.

We used two treatment modalities with 10 μM PLX5622 to deplete microglia: i) immediately after brain homogenization and ii) at day in vitro 12. The application of the inhibitor immediately after cell preparation depleted microglia to 8% at 1 week, to 2% at 4 weeks and to 0.5% at 6 weeks (half-time 3.5 days). When mixed glial cultures were treated starting at day in vitro 12, microglia depletion was slower (half-time 6 days) and not complete, indicating a decreased sensitivity to CSF-1. The remaining astrocytes preserved their proliferation ability, their migration in a scratch wound assay, and their pro-inflammatory (IL-6) response towards lipopolysaccharide.

The proposed approach for microglial depletion in mixed glial cultures is more effective than other existing methods and is non-toxic to non-microglial cells.

CSF-1R inhibitors are effective tools for depleting microglia in mixed glial cultures. Longer maturation of the cultures leads to a diminished sensitivity of microglia towards CSF-1. Thus, the treatment should start as early as possible after glial culture preparation.