Intermediate tissue oxygen level is required to observe murine FLASH skin sparing

Hunter, D. I.; Sunnerberg, J. P.; Tavakkoli, A. D.; Sloop, A. M.; Allen, B.; Gui, J.; Cao, X.; Zhang, R.; Swartz, H. M.; Gladstone, D. J.; Jarvis, L. A.; Hoopes, P. J.; Pogue, B. W.

doi:10.1101/2025.10.06.680759

bioRxiv · 10.1101/2025.10.06.680759

Intermediate tissue oxygen level is required to observe murine FLASH skin sparing

Hunter, D. I.·Sunnerberg, J. P.·Tavakkoli, A. D.·Sloop, A. M.·Allen, B.·Gui, J.·Cao, X.·Zhang, R.·Swartz, H. M.·Gladstone, D. J.·Jarvis, L. A.·Hoopes, P. J.·Pogue, B. W.

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Abstract

ObjectiveThis study evaluated the hypothesis that baseline tissue oxygen (pO2) would modulate FLASH toxicity sparing in murine skin, using a wide range of pO2 values, with ultra-high dose rate (UHDR) versus conventional dose rate (CDR) irradiation. ApproachMurine leg tissue pO2 was systematically varied and measured during irradiation from a FLASH Mobetron 9 MeV linac at 25 Gy, comparing UHDR ({approx}240 Gy/s) to CDR ({approx}0.16 Gy/s), for radiation induced skin toxicity outcomes. Baseline tissue pO2 was systematically modulated in 5 different treatment cohorts, using different ranges of inhaled gas (room air, 100% oxygen, or carbogen) and through varying limb vascular compression (partial or full). Radiolytic oxygen consumption, gO2 (mmHg/Gy), was quantified in vivo, and induced macroscopic skin toxicity was scored daily post treatment. Main ResultsFLASH skin sparing was observed at a fixed dose of 25 Gy in groups with partial leg clamping (pO2{approx}7{+/-}4mmHg), inhaled air (pO2{approx}12{+/-}6mmHg) and 100% oxygen (pO2{approx}16{+/-}4mmHg), while reduction in ulceration progression was significant only in the air inhalation group. No FLASH effect was observed under anoxic conditions, via complete blood flow occlusion (pO2{approx}0{+/-}1mmHg), or when modulated by inhaled carbogen (pO2{approx}21{+/-}7mmHg). In vivo measurements of radiolytic oxygen consumption, gO2, correlated to initial pO2 under UHDR conditions (pO2{approx}4-16mmHg), with ulceration predominantly occurring at pO2 values above 16mmHg. Inspired carbogen induced the highest pO2 at which point there was no FLASH sparing, for any dose groups between 25 to 15 Gy, despite having large changes in damage with dose. At the specific dose level of 25 Gy studied, the toxicity scores under anoxia for both UHDR and CDR were low (toxicity scores < 1) with no differences observed. SignificanceThese findings point to the fact that moderate and low tissue pO2 is associated with diminished oxygen-mediated damage at UHDR but not CDR, seen with inspired room air or 100% oxygen. Anoxic and hyperoxic murine skin are associated with minimal and maximal radiation damage respectively, but also exhibit no apparent FLASH toxicity sparing effect, with further investigation warranted into if the FLASH toxicity sparing effect persists at higher doses under anoxia.

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Hunter, D. I., Sunnerberg, J. P., Tavakkoli, A. D., Sloop, A. M., Allen, B., Gui, J., Cao, X., Zhang, R., Swartz, H. M., Gladstone, D. J., Jarvis, L. A., Hoopes, P. J., Pogue, B. W.. 2025-10-07. Intermediate tissue oxygen level is required to observe murine FLASH skin sparing. https://doi.org/10.1101/2025.10.06.680759

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