Timescale of FLASH Sparing Effect Determined by Varying Temporal Split of Dose Delivery in Mice

To determine the timescale for ultra high dose rate (UHDR) radiation delivery that dictates FLASH normal tissue sparing and elucidate its relationship to in vivo oxygen dynamics. A split-dose experiment was used to determine the transition time below which the observation of the FLASH sparing effect is preserved.

A 25 Gy dose was split into 2 deliveries (12.5 Gy), with varied interruption times. Albino B6 mice received flank skin irradiation in 8 groups: single-beam UHDR (25 Gy at 415 Gy/s), single-beam conventional dose rate (CDR) (25 Gy at 0.15 Gy/s), or split-beam delivery with 2 lower-dose UHDR beams (12.5 Gy at 415 Gy/s) separated by 0.1, 1, 5, 15, 25, or 120 seconds. Skin damage was scored daily for 31 days, with mixed-effects analysis comparing damage progression across cohorts. Real-time tissue pO₂ was monitored using the phosphorescence-lifetime probe, Oxyphor PdG4. Radiolytic oxygen consumption per unit dose (g_O2) and reoxygenation rates were quantified.

Single-beam UHDR significantly spared skin versus CDR. In split-dose groups, this sparing effect showed a transition at longer interbeam intervals. Damage progression remained significantly lower than CDR and comparable to single-beam UHDR (P > 0.16) for interruptions <15 seconds. Longer intervals progressively lost tissue sparing. Oximetry indicated an average tissue reoxygenation lifetime of 7.7 ± 1.1 seconds. At the delivery of the second beam, pO₂ remained lower when interbeam times were shorter than the reoxygenation period, but recovered fully for longer interruptions. The g_O2 values correlated with baseline tissue pO₂.

Observation of the FLASH sparing effect requires delivery within a critical temporal window that is similar to the timescale of tissue reoxygenation kinetics. The transition time for loss of the FLASH sparing effect in skin roughly corresponds to a diffusion timescale for oxygen, from capillaries to the cells. Although not conclusively demonstrating a mechanism, this unique finding supports the likelihood that local oxygen depletion or consumption underlies the FLASH tissue sparing effect observed in vivo, with important implications for clinical implementation and the timescale needed for multibeam FLASH radiation therapy.