Here, we highlight the potential translational benefits of delivering FLASH radiotherapy using ultra-high dose rates (>100 Gy·s −1 ). Compared with conventional dose-rate (CONV; 0.07-0.1 Gy·s −1 ) modalities, we showed that FLASH did not cause radiation-induced deficits in learning and memory in mice. Moreover, 6 months after exposure, CONV caused permanent alterations in neurocognitive end points, whereas FLASH did not induce behaviors characteristic of anxiety and depression and did not impair extinction memory. Mechanistic investigations showed that increasing the oxygen tension in the brain through carbogen breathing reversed the neuroprotective effects of FLASH, while radiochemical studies confirmed that FLASH produced lower levels of the toxic reactive oxygen species hydrogen peroxide. In addition, FLASH did not induce neuroinflammation, a process described as oxidative stress-dependent, and was also associated with a marked preservation of neuronal morphology and dendritic spine density. The remarkable normal tissue sparing afforded by FLASH may someday provide heretofore unrealized opportunities for dose escalation to the tumor bed, capabilities that promise to hasten the translation of this groundbreaking irradiation modality into clinical practice.ultra-high dose-rate irradiation | cognitive dysfunction | neuronal morphology | neuroinflammation | reactive oxygen species R adiation therapy (RT) remains an essential part of cancer treatment, and, today, the benefit of RT would increase dramatically if normal tissues surrounding the tumor could tolerate higher doses of radiation (1-3). In the last decade, major advances in high-precision treatment delivery and multimodal imaging have improved tolerance to RT (4), but the selective protection of normal tissue remains a significant clinical challenge and the radiation-induced toxicities still adversely impact the patient's quality of life. This latter fact largely remains an unmet medical need, and points to the urgency of developing improved RT modalities for combating those cancers refractory to treatment.This issue is especially critical for those afflicted with brain tumors, including glioblastoma multiforme (GBM), for which standard treatment consists of surgical resection followed by RT and concomitant chemotherapy (temozolomide). Typical radiotherapeutic protocols for GBM induce neurocognitive complications, including impairments in learning and memory, attention, and executive function and a variety of mood disorders (5-8). A breadth of past work from our laboratories has linked adverse neurocognitive outcomes following cranial irradiation to a range of neuropathologies, including reductions in dendritic complexity and spine density (9-12), reductions in microvascular density (13-15), reduced myelination and synapse density, and increased neuroinflammation (16,17). These changes are persistent and problematic in the conventionally irradiated brain and have prompted efforts to more fully develop a truly innovative approach to RT, where we have concept...

Cranial irradiation for the treatment of brain cancer elicits progressive and severe cognitive dysfunction that is associated with significant neuropathology. Radiation injury in the CNS has been linked to persistent microglial activation, and we find upregulation of pro-inflammatory genes even 6 weeks after irradiation. We hypothesize that depletion of microglia in the irradiated brain would have a neuroprotective effect. Adult mice received acute head only irradiation (9 Gy) and were administered a dietary inhibitor (PLX5622) of colony stimulating factor-1 receptor (CSF1R) to deplete microglia post-irradiation. Cohorts of mice maintained on a normal and PLX5662 diet were analyzed for cognitive changes using a battery of behavioral tasks 4–6 weeks later. PLX5622 treatment caused a rapid and near complete elimination of microglia in the brain within 3 days of treatment. Irradiation of animals given a normal diet caused characteristic behavioral deficits designed to test medial pre-frontal cortex (mPFC) and hippocampal learning and memory and caused increased microglial activation. Animals receiving the PLX5622 diet exhibited no radiation-induced cognitive deficits, and exhibited near complete loss of IBA-1 and CD68 positive microglia in the mPFC and hippocampus. Our data demonstrate that elimination of microglia through CSF1R inhibition can ameliorate radiation-induced cognitive deficits in mice.

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Cancer survivors face a variety of challenges as they cope with disease recurrence and a myriad of normal tissue complications brought on by radio-and chemotherapeutic treatment regimens. For patients subjected to cranial irradiation for the control of CNS malignancy, progressive and debilitating cognitive dysfunction remains a pressing unmet medical need. Although this problem has been recognized for decades, few if any satisfactory long-term solutions exist to resolve this serious unintended side effect of radiotherapy. Past work from our laboratory has demonstrated the neurocognitive benefits of human neural stem cell (hNSC) grafting in the irradiated brain, where intrahippocampal transplantation of hNSC ameliorated radiation-induced cognitive deficits. Using a similar strategy, we now provide, to our knowledge, the first evidence that cranial grafting of microvesicles secreted from hNSC affords similar neuroprotective phenotypes after headonly irradiation. Cortical-and hippocampal-based deficits found 1 mo after irradiation were completely resolved in animals cranially grafted with microvesicles. Microvesicle treatment was found to attenuate neuroinflammation and preserve host neuronal morphology in distinct regions of the brain. These data suggest that the neuroprotective properties of microvesicles act through a trophic support mechanism that reduces inflammation and preserves the structural integrity of the irradiated microenvironment.radiation-induced cognitive dysfunction | microvesicles | dendritic complexity | human neural stem cells | neuroinflammation W ith improved diagnosis and treatment, cancer survivorship continues to rise but often at the cost of quality of life. The unintended neurocognitive sequelae resulting from cranial irradiation used to treat primary and secondary malignancies of the brain are both progressive and debilitating (1, 2). Despite the recognition and prevalence of these adverse side effects, relatively few, if any, long-term satisfactory solutions exist for this unmet medical need. Past work from our laboratory has optimized transplantation parameters and established many of the long-term benefits of human stem cell-based therapies for the treatment of radiationinduced cognitive dysfunction (3-5). Cranially grafted stem cells have been shown to impart persistent improvements in behavioral performance in irradiated rats over extended postirradiation intervals (1-8 mo) using short-and long-term cognitive testing paradigms (4, 6, 7). These studies have shown that our stem cell-based approaches improve the functional plasticity of the host brain through a variety of mechanisms including (i) the suppression of neuroinflammation (5), (ii) the addition of new cells to active hippocampal circuits (4), and (iii) a long-term trophic support mechanism that facilitates the expression of activity-regulated cytoskeletonassociated protein that functions in multiple ways as a molecular determinant of memory (7). Moreover, using a distinctly different injury paradigm, stem cell grafting pres...

The present study examines functional contributions of microglia in host defense, demyelination, and remyelination following infection of susceptible mice with a neurotropic coronavirus. Treatment with PLX5622, an inhibitor of colony stimulating factor 1 receptor (CSF1R) that efficiently depletes microglia, prior to infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality compared with control mice that correlated with impaired control of viral replication. Single cell RNA sequencing (scRNASeq) of CD45+ cells isolated from the CNS revealed that PLX5622 treatment resulted in muted CD4+ T cell activation profile that was associated with decreased expression of transcripts encoding MHC class II and CD86 in macrophages but not dendritic cells. Evaluation of spinal cord demyelination revealed a marked increase in white matter damage in PLX5622‐treated mice that corresponded with elevated expression of transcripts encoding disease‐associated proteins Osteopontin (Spp1), Apolipoprotein E (Apoe), and Triggering receptor expressed on myeloid cells 2 (Trem2) that were enriched within macrophages. In addition, PLX5622 treatment dampened expression of Cystatin F (Cst7), Insulin growth factor 1 (Igf1), and lipoprotein lipase (Lpl) within macrophage populations which have been implicated in promoting repair of damaged nerve tissue and this was associated with impaired remyelination. Collectively, these findings argue that microglia tailor the CNS microenvironment to enhance control of coronavirus replication as well as dampen the severity of demyelination and influence repair.