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.

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.

FLASH irradiation protects synaptic density and spine morphology, 6 months after irradiation. A, Representative images of synaptophysin (red), DAPI (blue; Scale bar = 100 μm). B, Representative image of Homer1a (red)/Bassoon (green), respectively (Scale bar = 10 μm and 2 μm in the zoomed image). C and D, Quantification of synaptic density using synaptophysin found that FLASH did not induce dendritic disruptions that were observed in mice exposed to CONV-RT in both the Hippocampus and mPFC. E and F, Quantification of Homer1a and Bassoon spots within 120 nm of each other. Male and female mice exhibited no differences between presynaptic and postsynaptic binding after FLASH or CONV irradiation in the hippocampus or mPFC. 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 ≤ 0.001; ****, P ≤ 0.0001; ns, no significance.

Timeline. Ten-week-old mice received hypofractionated FLASH or CONV irradiation (3 × 10 Gy). A, Male and female mice underwent behavioral testing 4 months after irradiation. At 6 months post-irradiation, mice were sampled, and tissues were prepared for either IHC analysis (n = 4/sex/treatment) or ELISA (n = 5–8/sex/treatment). B, Female mice were used to assess LTP. At 4 months post-irradiation, animals performed in the extended NOR behavioral assay (n = 16/treatment). At 6 months post-irradiation, mice were sacrificed and prepared for electrophysiological analysis of long-term potentiation (n = 10–11/treatment).

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 protects synaptic density and spine morphology, 6 months after irradiation. A, Representative images of synaptophysin (red), DAPI (blue; Scale bar = 100 μm). B, Representative image of Homer1a (red)/Bassoon (green), respectively (Scale bar = 10 μm and 2 μm in the zoomed image). C and D, Quantification of synaptic density using synaptophysin found that FLASH did not induce dendritic disruptions that were observed in mice exposed to CONV-RT in both the Hippocampus and mPFC. E and F, Quantification of Homer1a and Bassoon spots within 120 nm of each other. Male and female mice exhibited no differences between presynaptic and postsynaptic binding after FLASH or CONV irradiation in the hippocampus or mPFC. 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 ≤ 0.001; ****, P ≤ 0.0001; ns, no significance.