In Vivo Imaging of the Microglial Landscape After Whole Brain Radiation Therapy

Abstract

Whole brain radiation therapy (WBRT) is an important treatment for patients with multiple brain metastases, but can also cause cognitive deterioration. Microglia, the resident immune cells of the brain, promote a proinflammatory environment and likely contribute to cognitive decline after WBRT. To investigate the temporal dynamics of the microglial reaction in individual mice to WBRT, we developed a novel in vivo experimental model using cranial window implants and longitudinal imaging. Chronic cranial windows were surgically implanted over the somatosensory cortex of transgenic Cx3cr1-enhanced green fluorescent protein (EGFP)/+ C57BL/6 mice, where microglia were fluorescently tagged with EGFP. Cx3cr1-EGFP/+ mice were also crossed with Thy1-YFP mice to fluorescently dual label microglia and subsets of neurons throughout the brain. Three weeks after window implantation and recovery, computed tomography image guided WBRT was delivered (single dose 10 Gy using two 5 Gy parallel-opposed lateral beams). Radiation dosing was confirmed using radiochromic film. Then, in vivo 2-photon microscopy was used to longitudinally image the microglial landscape and microglial motility at 7 days and 16 days after irradiation in the same mice. Film dosimetry confirmed the average delivered dose per beam at midpoint was accurate within 2%, with no attenuation from the window frame. By 7 days after WBRT, significant changes in the microglial landscape were seen, characterized by apparent loss of microglial cells (20%) and significant rearrangements of microglial location with time after irradiation (36% of cells not found in original location). Using longitudinal in vivo 2-photon imaging, this study demonstrated the feasibility of imaging microglia-neuron interactions and defining how microglia react to WBRT in the same mouse. Having demonstrated utility of the model, this experimental paradigm can be used to investigate the dynamic changes of many different brain cell types and their interactions after WBRT and uncover the underlying cellular mechanisms of WBRT-induced cognitive decline.