Single cell RNA-seq activated microglia cells after irradiation (IR)

Cranial irradiation (IR), an effective tool to treat malignant brain tumors, triggers a chronic pro-inflammatory microglial response, at least in the adult brain. Using single cell and bulk RNA sequencing combined with histology, we show that the microglial response in the juvenile mouse hippocampus is rapid, but returns toward normal within 1 week. We found that a single microglial cell simultaneously upregulates transcripts associated with pro- and anti-inflammatory microglial phenotypes. Our results indicate that microglia are involved in the initial stages, but may not be responsible for driving long-term inflammation in the juvenile brain. We performed whole brain irradiation on 108 postnatal day 21 (P21) with a single dose of 8 Gy. Using the LQ model and an α/β ratio of three for late effects in normal brain tissue, an acute exposure of 8 Gy is equivalent to approximately 18 Gy when delivered in repeated 2 Gy fractions, as in a clinical setting. Subsequently, animals were sacrificed at different time points starting from the acute phase after IR (2 h) up to later stages when hippocampal neurogenesis is severely diminished (6 weeks) compared to sham controls (SH). In order to investigate genes were expressed simultaneously in the same MG or whether hippocampal MG populations are phenotypically heterogeneous,we performed single-cell RNA-seq using the droplet-based method (10x Genomics Chromium) on 8,095 MG isolated at 6 h and 1 wk post-IR.