Purpose: FLASH radiation with ultra-high dose rate delivery is less damaging to normal tissue than conventional radiation ( <1 Gy/s). Since radiation depletes oxygen (ROD), this damage reduction might occur via the oxygen effect. ROD experiments have shown an oxygen-independent reduction in dose effectiveness at FLASH dose rates. However, prior in vivo ROD measurements relied on extracellular oxygen probes that could not penetrate cell membranes, leaving intracellular effects unresolved. To investigate the ROD hypothesis more directly, we developed a novel three-component immunohistochemical assay with algorithmic image processing to quantitatively compare DNA damage following FLASH and conventional irradiation in mouse jejunum. Methods: Mice received intravenous EF5 2 hours before proton irradiation at FLASH (103.63 +/- 17.2 Gy/s) or conventional (0.73 +/- 0.1 Gy/s) dose rates of 2.5 Gy or 5 Gy, with unirradiated controls. Mice were euthanized 30 minutes post-irradiation, and 10 cm of jejunum was frozen as a 'Swiss Roll', sectioned, stained, and imaged. Tissue sections were stained for {gamma}-H2AX, DRAQ5, and EF5 to assess DNA double-strand breaks, total DNA content, and hypoxia, respectively. An in-house algorithm identified individual cell nuclei and registered each nucleus with its corresponding {gamma}-H2AX and EF5 signals, enabling quantitative measurement of DNA damage as a function of local tissue hypoxia. Results: Hypoxia was greatest in the villi and, to a lesser extent, the outer jejunal musculature, with substantial inter-animal variation. DNA damage decreased in hypoxic regions. FLASH enhanced the hypoxia-associated reduction in DNA damage compared with conventional dose rate and, separately, revealed an oxygen-independent reduction in DNA damage, suggesting an additional FLASH sparing mechanism. Conclusion: Current results suggest that FLASH compared to conventional dose rate radiation caused less DNA damage with increasing effect at low oxygen levels, a result consistent with ROD as a mechanism. Pronounced tissue heterogeneity in murine jejunum requires further studies to segment the effect for each tissue type.
Curtis, N., Kim, M. M., Verginadis, I., Zou, W., Diffenderfer, E. S., Koumenis, C., Koch, C. J., Wiersma, R. D.
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