Implementation of ultra-high dose-rate FLASH radiotherapy (FLASH-RT) is rapidly gaining traction as a unique cancer treatment modality able to dramatically minimize normal tissue toxicity while maintaining antitumor efficacy compared with standard-of-care radiotherapy at conventional dose rate (CONV-RT). The resultant improvements in the therapeutic index have sparked intense investigations in pursuit of the underlying mechanisms. As a preamble to clinical translation, we exposed non–tumor-bearing male and female mice to hypofractionated (3 × 10 Gy) whole brain FLASH- and CONV-RT to evaluate differential neurologic responses using a comprehensive panel of functional and molecular outcomes over a 6-month follow-up. In each instance, extensive and rigorous behavioral testing showed FLASH-RT to preserve cognitive indices of learning and memory that corresponded to a similar protection of synaptic plasticity as measured by long-term potentiation (LTP). These beneficial functional outcomes were not found after CONV-RT and were linked to a preservation of synaptic integrity at the molecular (synaptophysin) level and to reductions in neuroinflammation (CD68+ microglia) throughout specific brain regions known to be engaged by our selected cognitive tasks (hippocampus, medial prefrontal cortex). Ultrastructural changes in presynaptic/postsynaptic bouton (Bassoon/Homer-1 puncta) within these same regions of the brain were not found to differ in response to dose rate. With this clinically relevant dosing regimen, we provide a mechanistic blueprint from synapse to cognition detailing how FLASH-RT reduces normal tissue complications in the irradiated brain. Significance: Functional preservation of cognition and LTP after hypofractionated FLASH-RT are linked to a protection of synaptic integrity and a reduction in neuroinflammation over protracted after irradiation times.

Irradiation parameters. Fractionated whole brain irradiation was performed on the Oriatron 6e, 6-MeV electron beam LINAC at Lausanne University Hospital. Mice received three doses of 10 Gy separated by 48 hours using a 17 mm graphite applicator at either CONV (CONV-RT; 0.09 Gy/second) or ultra-high dose-rate FLASH (FLASH-RT, delivered at 5.6 × 106 Gy/second in a single 1.8 μs pulse)

FLASH irradiation protects against reductions in LTP after CONV irradiation, 6 months after irradiation. A, Extended novel object recognition testing, 4 months after irradiation. Female mice that exposed to FLASH-RT performed significantly better than those who received CONV-RT (n = 15–16/treatment). B, TBS applied to the Schaffer collaterals produced a robust increase in fEPSP slope (as percent of baseline) in control and FLASH irradiated female mice but reduced in CONV mice 6 months after exposure. C, Levels of potentiation in the fEPSP slope maintained 1 hour after TBS was reduced significantly in the hippocampus of CONV-RT mice, but not in control or FLASH irradiated mice. Data were analyzed using a one-way ANOVA followed by Bonferroni multiple comparison test (n = 10–11/treatment). *, P ≤ 0.05: ***, P ≤ 0.001; ****, P ≤ 0.0001.

Mice exposed to FLASH-RT performed similar to controls in the NOR, LDB, and FE tests. A, NOR testing. Male mice exposed to FLASH-RT performed similar to controls, while CONV-RT were unable to differentiate between the familiar and novel object. Female mice exposed to FLASH-RT and CONV-RT exhibited no significant difference between controls. B, Measurement of transition between light and dark environments in LDB testing. Male and female mice exposed to CONV-RT performed significantly worse than controls. Male FLASH-RT mice were not significantly different than controls; however, female mice did not transition between arenas as controls did. C and D, Fear extinction training and extinction days. Exposure to CONV-RT caused male mice to exhibit increased freezing during training, while this was not observed in FLASH-RT or females. Exposure to CONV-RT also inhibited mice ability disassociate the tone/shock pairing as well as FLASH and controls in males and females. Group effects (#) were found in training days indicate that CONV irradiated animals exhibited increase freezing behavior. E and F, Fear extinction testing. FLASH and control mice greatly reduced their tone/shock associations while CONV male (left) and female (right) mice did not. Data were analyzed using a one-way or two-way ANOVA followed by Bonferroni multiple comparison test (n = 11–15/sex/treatment). *, P ≤ 0.05; **, P ≤ 0.01; #, P ≤ 0.05; ns, no significance.

FLASH irradiation protected against prolonged inflammation found in CONV mice, 6 months after irradiation. Representative images of reactive microglia CD68 (red) and DAPI (blue) in the male mouse hippocampus (A) and representative images of IBA1 (green), TLR4 (red), and DAPI (blue; Scale bar = 100 μm; B). C, Quantification of CD68 immunofluorescence in the hippocampus and medial prefrontal cortex. Male (left) and female (right) mice exposed to FLASH-RT exhibit no significant change in CD68 expression while CONV mice expressed a neuroinflammatory response. D, Quantification of IBA1 and TLR4 colocalization in the hippocampus and medial prefrontal cortex. Male and female mice exhibited decreased levels of the neuroinflammatory mediator TLR4 when compared with CONV irradiation. E, Inflammatory cytokines measured using ELISA. IL1α exhibited elevated expression after CONV-RT exposure when compared with controls while FLASH induced no changes. No significant changes were observed in TNFα or IL1β. All data were analyzed using a one-way ANOVA followed by Bonferroni multiple comparison test (n = 4/sex/treatment, two sections analyzed/region/animal). *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001.

Implementation of ultra-high dose-rate FLASH radiotherapy (FLASH-RT) is rapidly gaining traction as a unique cancer treatment modality able to dramatically minimize normal tissue toxicity while maintaining antitumor efficacy compared with standard-of-care radiotherapy at conventional dose rate (CONV-RT). The resultant improvements in the therapeutic index have sparked intense investigations in pursuit of the underlying mechanisms. As a preamble to clinical translation, we exposed non–tumor-bearing male and female mice to hypofractionated (3 × 10 Gy) whole brain FLASH- and CONV-RT to evaluate differential neurologic responses using a comprehensive panel of functional and molecular outcomes over a 6-month follow-up. In each instance, extensive and rigorous behavioral testing showed FLASH-RT to preserve cognitive indices of learning and memory that corresponded to a similar protection of synaptic plasticity as measured by long-term potentiation (LTP). These beneficial functional outcomes were not found after CONV-RT and were linked to a preservation of synaptic integrity at the molecular (synaptophysin) level and to reductions in neuroinflammation (CD68+ microglia) throughout specific brain regions known to be engaged by our selected cognitive tasks (hippocampus, medial prefrontal cortex). Ultrastructural changes in presynaptic/postsynaptic bouton (Bassoon/Homer-1 puncta) within these same regions of the brain were not found to differ in response to dose rate. With this clinically relevant dosing regimen, we provide a mechanistic blueprint from synapse to cognition detailing how FLASH-RT reduces normal tissue complications in the irradiated brain.Significance:Functional preservation of cognition and LTP after hypofractionated FLASH-RT are linked to a protection of synaptic integrity and a reduction in neuroinflammation over protracted after irradiation times.