Fig. 5: CSF1R inhibition regulates microglial cell turnover.

a Murine mixed glial cultures were treated with sCSF1R_inh and Iba1 immunocytochemistry was used to identify microglia after 3 days. Representative images from the INCell Analyzer Imaging system demonstrate a qualitative reduction in microglia following CSF1R inhibition. Scale bar: 100 µm. b Quantitative analysis of Iba1⁺ area reveals that sCSF1R_inh significantly depletes microglia in a concentration-dependent manner. Data points represent the mean and standard deviation (n = 3); the IC₅₀ was calculated with Prism 6 (GraphPad Software). c Murine mixed glial cultures pretreated with zVAD blocks sCSF1R_inh induced microglial cell death after 3 days. d Iba1 immunohistochemistry reveals a dose dependent decrease in the number of cortical microglia in naïve C57BL/6 mice treated with vehicle versus sCSF1R_inh for 7 days. Scale bar: 100 µm. Manual quantification of Iba1⁺ cells confirmed these qualitative observations. Data points represent the average Iba⁺ cell count per animal (n = 3 images/animal, n = 4 animals/group). Graphical columns represent the mean and standard error. Statistical significance was determined by a one-way ANOVA and p values are indicated by ***p < 0.001, ****p < 0.0001. e Primary murine microglia were treated with sCSF1R_inh and cell viability was assessed after 5 days utilizing Promega’s Cell Titer Glo 2.0 Assay Kit. sCSF1R_inh treatment had no impact on microglial cell survival at the concentrations assessed in this experiment. Data points represent the viability in each well and graphical columns represent the mean and standard deviation of three wells. f CSF1 levels were elevated in conditioned media from mixed murine glial cultures as well as primary microglia and astrocytes. Data points represent the protein concentration per well and graphical columns represent the mean and standard deviation of six wells. Statistical significance was determined by a one-way ANOVA performed and p values are indicated by **p < 0.01, ****p < 0.0001. g The A1 cocktail induced a concentration-dependent increase in CSF1 production by primary human astrocytes. Data points represent the protein concentration per well and graphical columns represent the mean and standard deviation of six wells. Statistical significance was determined by a one-way ANOVA performed and p values are indicated by **p < 0.01, ****p < 0.0001. h Pseudo bulk sequencing analysis of CSF1 expression in astrocytes from control versus MS patient from previously published CNS single nuclei sequencing data⁴⁶. i In situ hybridization with CSF1 probe and immunohistochemistry with a GFAP antibody reveals the presence of CSF1 transcripts within astrocytes in the progressive MS brain. Scale bar: 50 µm.