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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.

Background Ultrahigh dose-rate radiotherapy (FLASH-RT) affords improvements in the therapeutic index by minimizing normal tissue toxicities without compromising antitumor efficacy compared to conventional dose-rate radiotherapy (CONV-RT). To investigate the translational potential of FLASH-RT to a human pediatric medulloblastoma brain tumor, we used a radiosensitive juvenile mouse model to assess adverse long-term neurological outcomes. Methods Cohorts of 3-week-old male and female C57Bl/6 mice exposed to hypofractionated (2 × 10 Gy, FLASH-RT or CONV-RT) whole brain irradiation and unirradiated controls underwent behavioral testing to ascertain cognitive status four months posttreatment. Animals were sacrificed 6 months post-irradiation and tissues were analyzed for neurological and cerebrovascular decrements. Results The neurological impact of FLASH-RT was analyzed over a 6-month follow-up. FLASH-RT ameliorated neurocognitive decrements induced by CONV-RT and preserved synaptic plasticity and integrity at the electrophysiological (long-term potentiation), molecular (synaptophysin), and structural (Bassoon/Homer-1 bouton) levels in multiple brain regions. The benefits of FLASH-RT were also linked to reduced neuroinflammation (activated microglia) and the preservation of the cerebrovascular structure, by maintaining aquaporin-4 levels and minimizing microglia colocalized to vessels. Conclusions Hypofractionated FLASH-RT affords significant and long-term normal tissue protection in the radiosensitive juvenile mouse brain when compared to CONV-RT. The capability of FLASH-RT to preserve critical cognitive outcomes and electrophysiological properties over 6-months is noteworthy and highlights its potential for resolving long-standing complications faced by pediatric brain tumor survivors. While care must be exercised before clinical translation is realized, present findings document the marked benefits of FLASH-RT that extend from synapse to cognition and the microvasculature.

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 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.

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.

Mice exposed to FLASH-RT performed similar to controls in hippocampal-dependent learning and memory tests OUL while CONV-RT mice did not. A, Objects in updated locations testing experimental design. B, Update session behavior. At 4 months post-irradiation, FLASH and control mice showed preference for the novel toy and location in both males (left) and females (right) while CONV did not. C, Updated information test session. CONV irradiated female mice failed to learn the updated novel (A4) object over its predecessor (A3) when compared with FLASH and control mice. CONV irradiated male mice performed significantly worse than FLASH mice. D, Original information test session. CONV irradiated female mice were unable to differentiate between the updated novel location (A4) and the original location (A1) while FLASH and control performed similarly. No significant changes in male mice were observed. All data were analyzed using a one-way ANOVA followed by Bonferroni multiple comparison test (n = 11–16/sex/treatment). *, P ≤ 0.05; **, P ≤ 0.01; ns, no significance.

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)

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).

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.

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 anti-tumor efficacy compared to 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 (3x10 Gy) whole brain FLASH- and CONV-RT to evaluate differential neurological 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). Ultra-structural changes in pre/post-synaptic 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. Citation Format: Charles L. Limoli, Barrett D. Allen, Eniko A. Kramár, Yasaman Alaghband, Marie-Catherine Vozenin. Uncovering the protective neurological mechanisms of hypofractionated FLASH radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2416.

Technological advancements have facilitated the implementation of realistic, terrestrial-based complex 33-beam galactic cosmic radiation simulations (GCR Sim) to now probe central nervous system functionality. This work expands considerably on prior, simplified GCR simulations, yielding new insights into responses of male and female mice exposed to 40–50 cGy acute or chronic radiations relevant to deep space travel. Results of the object in updated location task suggested that exposure to acute or chronic GCR Sim induced persistent impairments in hippocampus-dependent memory formation and reconsolidation in female mice that did not manifest robustly in irradiated male mice. Interestingly, irradiated male mice, but not females, were impaired in novel object recognition and chronically irradiated males exhibited increased aggressive behavior on the tube dominance test. Electrophysiology studies used to evaluate synaptic plasticity in the hippocampal CA1 region revealed significant reductions in long-term potentiation after each irradiation paradigm in both sexes. Interestingly, network-level disruptions did not translate to altered intrinsic electrophysiological properties of CA1 pyramidal cells, whereas acute exposures caused modest drops in excitatory synaptic signaling in males. Ultrastructural analyses of CA1 synapses found smaller postsynaptic densities in larger spines of chronically exposed mice compared to controls and acutely exposed mice. Myelination was also affected by GCR Sim with acutely exposed mice exhibiting an increase in the percent of myelinated axons; however, the myelin sheathes on small calibur ( 0.5 mm) axons were thinner when compared to controls. Present findings might have been predicted based on previous studies using single and mixed beam exposures and provide further evidence that space-relevant radiation exposures disrupt critical cognitive processes and underlying neuronal network-level plasticity, albeit not to the extent that might have been previously predicted.