Major advances in high precision treatment delivery and imaging have greatly improved the tolerance of radiotherapy (RT); however, the selective sparing of normal tissue and the reduction of neurocognitive side effects from radiation-induced toxicities remain significant problems for pediatric patients with brain tumors. While the overall survival of pediatric patients afflicted with medulloblastoma (MB), the most common type primary brain cancer in children, remains high (≥80%), lifelong neurotoxic side-effects are commonplace and adversely impact patients’ quality of life. To circumvent these clinical complications, we have investigated the capability of ultra-high dose rate FLASH-radiotherapy (FLASH-RT) to protect the radiosensitive juvenile mouse brain from normal tissue toxicities. Compared to conventional dose rate (CONV) irradiation, FLASH-RT was found to ameliorate radiation-induced cognitive dysfunction in multiple independent behavioral paradigms, preserve developing and mature neurons, minimize microgliosis and limit the reduction of the plasmatic level of growth hormone. The protective “FLASH effect” was pronounced, especially since a similar whole brain dose of 8 Gy delivered with CONV-RT caused marked reductions in multiple indices of behavioral performance (objects in updated location, novel object recognition, fear extinction, light-dark box, social interaction), reductions in the number of immature (doublecortin⁺) and mature (NeuN⁺) neurons and increased neuroinflammation, adverse effects that were not found with FLASH-RT. Our data point to a potentially innovative treatment modality that is able to spare, if not prevent, many of the side effects associated with long-term treatment that disrupt the long-term cognitive and emotional well-being of medulloblastoma survivors.

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.

A recognized risk of long-duration space travel arises from the elevated exposure astronauts face from galactic cosmic radiation (GCR), which is composed of a diverse array of energetic particles. There is now abundant evidence that exposures to many different charged particle GCR components within acute time frames are sufficient to induce central nervous system deficits that span from the molecular to the whole animal behavioral scale. Enhanced spacecraft shielding can lessen exposures to charged particle GCR components, but may conversely elevate neutron radiation levels. We previously observed that space-relevant neutron radiation doses, chronically delivered at dose-rates expected during planned human exploratory missions, can disrupt hippocampal neuronal excitability, perturb network long-term potentiation and negatively impact cognitive behavior. We have now determined that acute exposures to similar low doses (18 cGy) of neutron radiation can also lead to suppressed hippocampal synaptic signaling, as well as decreased learning and memory performance in male mice. Our results demonstrate that similar nervous system hazards arise from neutron irradiation regardless of the exposure time course. While not always in an identical manner, neutron irradiation disrupts many of the same central nervous system elements as acute charged particle GCR exposures. The risks arising from neutron irradiation are therefore important to consider when determining the overall hazards astronauts will face from the space radiation environment.

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).