Time-dependent alterations in brain metabolites and gut microbiota following whole-brain FLASH versus conventional radiotherapy in mice

PURPOSE: This study compared the time-dependent changes in brain metabolites and gut microbiota at early and late post-irradiation stages in mice receiving conventional radiotherapy (CONV-RT) or ultra-high dose-rate FLASH radiotherapy (FLASH-RT). METHODS: Male C57BL/6 J mice received whole-brain irradiation using CONV-RT (2 Gy/min) or X-ray-based FLASH-RT (200 Gy/s). Brain tissue and gut contents were collected on days 1, 3, 7, and 21 post-irradiation. Targeted LC-MS metabolomics profiled dynamic brain metabolite changes, and 16S rRNA sequencing characterized gut microbiota trajectories. Neuroinflammation and brain injury were assessed by immunofluorescence (microglia/astrocyte markers) and laser speckle cerebral blood flow imaging, while behavioral assays evaluated cognition and anxiety-like behaviors. RESULTS: Compared with CONV-RT, FLASH-RT was associated with less pronounced hippocampal microglial and astrocytic reactivity, a smaller reduction in cerebral blood flow perfusion, and attenuated radiation-associated cognitive deficits. Metabolomics revealed distinct temporal trajectories: CONV-RT showed sustained late-phase suppression of metabolic programs, whereas FLASH-RT exhibited a coordinated late-stage rebound in pathways related to amino acid/carbohydrate metabolism and synaptic neurotransmission. Mechanistically relevant metabolites linked to antioxidation, energy metabolism, and neural repair were increased under FLASH-RT, consistent with reduced neuroinflammation. Gut microbiota profiling demonstrated that FLASH-RT induced a milder and more transient dysbiosis, with faster restoration toward a Sham-like structure and enrichment of putative beneficial taxa (including Lachnospiraceae, Peptostreptococcaceae, and Dubosiella), while CONV-RT was associated with more persistent Proteobacteria/Enterobacteriaceae-related disruptions and opportunistic signatures. CONCLUSION: In summary, we have innovatively explored the changes in brain metabolites and gut microbiota induced by FLASH-RT whole-brain irradiation, providing a theoretical foundation for further investigation into the mechanisms underlying FLASH-RT's effects.