Sabrina Hupp1, Asparouh I Iliev2. 1. Institute of Anatomy, University of Bern, Baltzerstrasse 2, 3012, Bern, Switzerland. Electronic address: sabrina.hupp@ana.unibe.ch. 2. Institute of Anatomy, University of Bern, Baltzerstrasse 2, 3012, Bern, Switzerland. Electronic address: asparouh.iliev@ana.unibe.ch.
Abstract
BACKGROUND: 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. NEW METHOD: 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. RESULTS: 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. COMPARISON TO EXISTING METHODS: 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. CONCLUSIONS: 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.
BACKGROUND: 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. NEW METHOD: 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. RESULTS: 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. COMPARISON TO EXISTING METHODS: 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. CONCLUSIONS: 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.
Authors: Line S Reinert; Søren R Paludan; Georgios Katzilieris-Petras; Xin Lai; Ahmad S Rashidi; Georges M G M Verjans Journal: J Virol Date: 2022-01-19 Impact factor: 6.549