CSF1R inhibition during cranial radiotherapy reshapes the immune environment and glial dynamics

Background: Cranial radiotherapy (cRT) induces long-lasting alterations in the brain microenvironment, including changes in glial populations and loss of neurogenesis. Pharmacological depletion of microglia via colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a strategy to modulate radiation-induced neuroinflammation, but its effects on other glial populations remains incompletely understood. Methods: Nine-week-old male C57BL/6JRj mice (n=60) received either a CSF1R-inhibitor supplemented diet (pexidartinib, PLX3397) or a control diet prior to and continued for 10 days after fractionated CT-guided cRT (3x10 Gy, 30 Gy). Brain tissue was assessed at multiple time-points up to six months post cRT using immunohistochemistry for markers of microglia, astrocytes, oligodendrocytes and proliferating cells. Microglial identity and repopulation dynamics were assessed using IBA1, F4/80 and the homeostatic microglial marker P2RY12, complemented by morphometric analysis. Results: Pexidartinib alone resulted in partial microglial depletion, whereas its combination with cRT led to near-complete loss of P2RY12+ homeostatic microglia at early time points. After discontinuation of the diet this was accompanied by increased IBA1+;F4/80high;P2RY12neg cell densities, consistent with replacement of the microglial niche by monocyte-derived cells. Over time, these cells exhibited gradual morphological adaptation but remained largely P2RY12neg, indicating incomplete restoration of a homeostatic microglial phenotype. Combined treatment accelerated astrocytic reactivity compared to cRT alone and did not prevent cRT-induced loss of neurogenesis or OPCs. At later time points, partial OPC recovery was observed in selected regions in the combined treatment group. Conclusion: CSF1R inhibition during cRT profoundly alters the composition and temporal dynamics of the brain its myeloid compartment, characterized by sustained loss of homeostatic microglia and replacement by monocyte-derived cells. Under these experimental conditions, microglial depletion did not mitigate key histopathological features associated with radiation-induced brain injury and was associated with accelerated astrocytic responses. These findings highlight the importance of timing, extent of depletion, and repopulation dynamics when considering microglia-targeting strategies in the context of cranial radiotherapy.