Response of primary astrocytes to ionizing radiation exposure

Abstract

Introduction: Exposure to space conditions during crewed long-term exploration missions can cause several health risks for astronauts. Space radiation, isolation and microgravity are major limiting factors. The role of astrocytes in cognitive disturbances by space radiation is unknown. Astrocytes’ response towards low linear energy transfer (LET) X-rays and high-LET carbon (¹²C) and iron (⁵⁶Fe) ions was compared to reveal possible effects of space-relevant high-LET radiation. Methods: Primary murine cortical astrocytes were irradiated with different doses of X-rays, ¹²C and ⁵⁶Fe ions at the heavy ion accelerator GSI. DNA damage and repair (γH2AX, 53BP1), cell proliferation (Ki-67), astrocytes’ reactivity (GFAP) and NF-κB pathway activation (p65) were analyzed by immunofluorescence microscopy. Cell cycle progression was investigated by flow cytometry of DNA content. Gene expression changes after exposure to X-rays were investigated by mRNA-sequencing. RT-qPCR for the genes of interest was performed with X-rays- and heavy-ion-irradiated astrocytes: Cdkn1a, Cdkn2a, Gfap, Tnf, Il1β, Il6 and Tgfβ1. Levels of the pro-inflammatory cytokine IL-6 were determined using ELISA. Results: Astrocytes showed distinct responses towards the three different radiation qualities. Induction of radiation-induced DNA double strand breaks (DSB) and the respective repair was dose-, LET- and time-dependent. Proliferation and cell cycle progression were not affected by radiation qualities examined in this study. Astrocytes expressed IL-6 and GFAP with constitutive NF-κB activity independent of radiation exposure. mRNA sequencing of X-irradiated astrocytes revealed downregulation of 66 genes involved in DNA damage response and repair, mitosis, proliferation and cell cycle regulation. Conclusion: Primary murine astrocytes are DNA repair proficient irrespective of radiation quality. Only minor gene expression changes were observed after X-ray exposure and reactivity was not induced. Co-culture of astrocytes with microglial cells, brain organoids or organotypic brain slice culture experiments might reveal whether astrocytes show a more pronounced radiation response in more complex network architectures in the presence of other neuronal cell types. Acknowledgement: We thank our liaison scientists at GSI, Insa Schröder and Denise Eckart, for their excellent technical assistance in preparation of and during the beamtimes. Ulrich Weber and Thomas Friedrich at GSI are acknowledged for their dedicated and precise irradiation of our samples at GSI. Our thanks also go to the beam operators at GSI for operating the accelerator during our experiments.