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The pervasiveness of deep space radiation remains a confounding factor for the transit of humans through our solar system. Spacecraft shielding both protects astronauts but also contributes to absorbed dose through galactic cosmic ray interactions that produce secondary particles. The resultant biological effects drop to a minimum for aluminum shielding around 20 g/cm2 but increase with additional shielding. The present work evaluates for the first time, the impact of secondary pions on central nervous system functionality. The fractional pion dose emanating from thicker shielded spacecraft regions could contribute up to 10% of the total absorbed radiation dose. New results from the Paul Scherrer Institute have revealed that low dose exposures to 150 MeV positive and negative pions, akin to a Mars mission, result in significant, long-lasting cognitive impairments. These surprising findings emphasize the need to carefully evaluate shielding configurations to optimize safe exposure limits for astronauts during deep space travel.

Astronauts will encounter extended exposure to galactic cosmic radiation (GCR) during deep space exploration, which could impair brain function. Here, we report that in male mice, acute or chronic GCR exposure did not modify reward sensitivity but did adversely affect attentional processes and increased reaction times. Potassium (K+)-stimulation in the prefrontal cortex (PFC) elevated dopamine (DA) but abolished temporal DA responsiveness after acute and chronic GCR exposure. Unlike acute GCR, chronic GCR increased levels of all other neurotransmitters, with differences evident between groups after higher K+-stimulation. Correlational and machine learning analysis showed that acute and chronic GCR exposure differentially reorganized the connection strength and causation of DA and other PFC neurotransmitter networks compared to controls which may explain space radiation-induced neurocognitive deficits.

BackgroundUltrahigh 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.MethodsCohorts 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.ResultsThe 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.ConclusionsHypofractionated 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.

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.SignificanceFunctional 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.

Traditionally, the brain has been regarded as a relatively insensitive late-reacting tissue, with radiologically detectable damage not being reported at doses < 60 Gy. When NASA proposed interplanetary exploration missions, it was required to conduct an intensive health and safety evaluation of cancer, cardiovascular, and cognitive risks associated with exposure to deep space radiation (SR). The SR dose that astronauts on a mission to Mars are predicted to receive is ~300 mGy. Even after correcting for the higher RBE of the SR particles, the biologically effective SR dose (

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.

The response of the human body to multiple spaceflight stressors is complex, but mounting evidence implicate risks to CNS functionality as significant, able to threaten metrics of mission success and longer-term behavioral and neurocognitive health. Prolonged exposure to microgravity, sleep disruption, social isolation, fluid shifts, and ionizing radiation have been shown to disrupt mechanisms of homeostasis and neurobiological well-being. The overarching goal of this review is to document the existing evidence of how the major spaceflight stressors, including radiation, microgravity, isolation/confinement, and sleep deprivation, alone or in combination alter molecular, neurochemical, neurobiological, and plasma metabolite/lipid signatures that may be linked to operationally-relevant behavioral and cognitive performance. While certain brain region-specific and/or systemic alterations titrated in part with neurobiological outcome, variations across model systems, study design, and the conspicuous absence of targeted studies implementing combinations of spaceflight stressors, confounded the identification of specific signatures having direct relevance to human activities in space. Summaries are provided for formulating new research directives and more predictive readouts of portending change in neurobiological function.

Current radiotherapy facilities are standardized to deliver dose rates around 0.1-0.4 Gy/s in 2 Gy daily fractions, designed to deliver total accumulated doses to reach the tolerance limit of normal tissues undergoing irradiation. FLASH radiotherapy (FLASH-RT), on the other hand, relies on facilities capable of delivering ultrahigh dose rates in large doses in a single microsecond pulse, or in a few pulses given over a very short time sequence. For example, most studies to date have implemented 4-6 MeV electrons with intra-pulse dose rates in the range 106 -107  Gy/s. The proposed dependence of the FLASH effect on oxygen tension has stimulated several theoretical models based on three different hypotheses: (i) Radiation-induced transient oxygen depletion; (ii) cell-specific differences in the ability to detoxify and/or recover from injury caused by reactive oxygen species; (iii) self-annihilation of radicals by bimolecular recombination. This article focuses on the observations supporting or refuting these models in the frame of the chemical-biological bases of the impact of oxygen on the radiation response of cell free, in vitro and in vivo model systems.

PurposeSpatially fractionated radiation therapy (SFRT), which delivers highly nonuniform dose distributions instead of conventionally practiced homogeneous tumor dose, has shown high rates of clinical response with minimal toxicities in large-volume primary or metastatic malignancies. However, prospective multi-institutional clinical trials in SFRT are lacking, and SFRT techniques and dose parameters remain variable. Agreement on dose prescription, technical administration, and clinical and translational design parameters for SFRT trials is essential to enable broad participation and successful accrual to rigorously test the SFRT approach. We aimed to develop a consensus for the design of multi-institutional clinical trials in SFRT, tailored to specific primary tumor sites, to help facilitate development and enhance the feasibility of such trials.Methods and materialsPrimary tumor sites with sufficient pilot experience in SFRT were identified, and fundamental trial design questions were determined. For each tumor site, a comprehensive consensus effort was established through disease-specific expert panels. Clinical trial design criteria included eligibility, SFRT technology and technique, dose and fractionation, target- and normal-tissue dose parameters, systemic therapies, clinical trial endpoints, and translational science considerations. Iterative appropriateness rank voting, expert panel consensus reviews and discussions, and public comment posting were used for consensus development.ResultsClinical trial criteria were developed for head and neck cancer and soft-tissue sarcoma. Final consensus among the 22 trial design categories each (a total of 163 criteria) was high to moderate overall. Uniform patient cohorts of advanced bulky disease, standardization of SFRT technologies and dosimetry and physics parameters, and collection of translational correlates were considered essential to trial design. Final guideline recommendations and the degree of agreement are presented and discussed.ConclusionsThis consensus provides design guidelines for the development of prospective multi-institutional clinical trials testing SFRT in advanced head and neck cancer and soft-tissue sarcoma through in-advance harmonization of the fundamental clinical trial design among SFRT experts, potential investigators, and the SFRT community.

Exposure to radiation during the treatment of CNS tumors leads to detrimental damage of the blood brain barrier (BBB) in normal tissue. Effects are characterized by leakage of the vasculature which exposes the brain to a host of neurotoxic agents potentially leading to white matter necrosis, parenchymal calcification, and an increased chance of stroke. Vasculature of the blood tumor barrier (BTB) is irregular leading to poorly perfused and hypoxic tissue throughout the tumor that becomes resistant to radiation. While current clinical applications of cranial radiotherapy use dose fractionation to reduce normal tissue damage, these treatments still cause significant alterations to the cells that make up the neurovascular unit of the BBB and BTB. Damage to the vasculature manifests as reduction in tight junction proteins, alterations to membrane transporters, impaired cell signaling, apoptosis, and cellular senescence. While radiotherapy treatments are detrimental to normal tissue, adapting combined strategies with radiation targeted to damage the BTB could aid in drug delivery. Understanding differences between the BBB and the BTB may provide valuable insight allowing clinicians to improve treatment outcomes. Leveraging this information should allow advances in the development of therapeutic modalities that will protect the normal tissue while simultaneously improving CNS tumor treatments.

The taxane family of microtubule poisons and chemotherapeutics have been studied for over 50 years and are among the most frequently used antineoplastic agents today. Still, limited research exists characterizing taxane-induced sex-specific mechanisms of action and toxicities in cancer and non-cancerous tissue. Such research is important to advance cancer treatment outcomes as well as to address clinically observed sex-differences in short- and long-term taxane-induced toxicities that have disproportionate effects on female and male cancer patients. To gain more insight into these underlying differences between the sexes, the following review draws from pre-clinical and clinical paclitaxel and taxane oncology literature, examines sex-discrepancies, and highlights uncharacterized sex-dependent mechanisms of action and clinical outcomes. To our knowledge, this is the first literature review to provide a current overview of the basic and clinical sex dimorphisms of taxane-induced effects. Most importantly, we hope to provide a starting point for improving and advancing sex-specific personalized chemotherapy and cancer treatment strategies as well as to present a novel approach to review sex as a biological variable in basic and clinical biology.

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.

Galactic cosmic radiation (GCR), composed of highly energetic and fully ionized atomic nuclei, produces diverse deleterious effects on the body. In researching the neurological risks of GCR exposures, including during human spaceflight, various ground-based single-ion GCR irradiation paradigms induce differential disruptions of cellular activity and overall behavior. However, it remains less clear how irradiation comprising a mix of multiple ions, more accurately recapitulating the space GCR environment, impacts the central nervous system. We therefore examined how mixed-ion GCR irradiation (two similar 5-6 beam combinations of protons, helium, oxygen, silicon and iron ions) influenced neuronal connectivity, functional generation of activity within neural circuits and cognitive behavior in mice. In electrophysiological recordings we find that space-relevant doses of mixed-ion GCR preferentially alter hippocampal inhibitory neurotransmission and produce related disruptions in the local field potentials of hippocampal oscillations. Such underlying perturbation in hippocampal network activity correspond with perturbed learning, memory and anxiety behavior.

The proposed deep space exploration to the moon and later to Mars will result in astronauts receiving significant chronic exposures to space radiation (SR). SR exposure results in multiple neurocognitive impairments. Recently, our cross-species (mouse/rat) studies reported impaired associative memory formation in both species following a chronic 6-month low dose exposure to a mixed field of neutrons (1 mGy/day for a total dose pf 18 cGy). In the present study, we report neutron exposure induced synaptic plasticity in the medial prefrontal cortex, accompanied by microglial activation and significant synaptic loss in the hippocampus. In a parallel study, neutron exposure was also found to alter fluorescence assisted single synaptosome LTP (FASS-LTP) in the hippocampus of rats, that may be related to a reduced ability to insert AMPAR into the post-synaptic membrane, which may arise from increased phosphorylation of the serine 845 residue of the GluA1 subunit. Thus, we demonstrate for the first time, that low dose chronic neutron irradiation impacts homeostatic synaptic plasticity in the hippocampal-cortical circuit in two rodent species, and that the ability to successfully encode associative recognition memory is a dynamic, multicircuit process, possibly involving compensatory changes in AMPAR density on the synaptic surface.

PurposeRecent data have shown that single-fraction irradiation delivered to the whole brain in less than tenths of a second using FLASH radiotherapy (FLASH-RT), does not elicit neurocognitive deficits in mice. This observation has important clinical implications for the management of invasive and treatment-resistant brain tumors that involves relatively large irradiation volumes with high cytotoxic doses.Experimental designTherefore, we aimed at simultaneously investigating the antitumor efficacy and neuroprotective benefits of FLASH-RT 1-month after exposure, using a well-characterized murine orthotopic glioblastoma model. As fractionated regimens of radiotherapy are the standard of care for glioblastoma treatment, we incorporated dose fractionation to simultaneously validate the neuroprotective effects and optimized tumor treatments with FLASH-RT.ResultsThe capability of FLASH-RT to minimize the induction of radiation-induced brain toxicities has been attributed to the reduction of reactive oxygen species, casting some concern that this might translate to a possible loss of antitumor efficacy. Our study shows that FLASH and CONV-RT are isoefficient in delaying glioblastoma growth for all tested regimens. Furthermore, only FLASH-RT was found to significantly spare radiation-induced cognitive deficits in learning and memory in tumor-bearing animals after the delivery of large neurotoxic single dose or hypofractionated regimens.ConclusionsThe present results show that FLASH-RT delivered with hypofractionated regimens is able to spare the normal brain from radiation-induced toxicities without compromising tumor cure. This exciting capability provides an initial framework for future clinical applications of FLASH-RT.See related commentary by Huang and Mendonca, p. 662.

Human stem cell-derived extracellular vesicles (EV) provide many advantages over cell-based therapies for the treatment of functionally compromised tissue beds and organ sites. Here we sought to determine whether human embryonic stem cell (hESC)-derived EV could resolve in part, the adverse late normal tissue complications associated with exposure of the lung to ionizing radiation. The hESC-derived EV were systemically administered to the mice via the retro-orbital sinus to explore the potential therapeutic benefits following exposure to high thoracic doses of radiation (14 Gy). Data demonstrated that hESC-derived EV treatment significantly improved overall survival of the irradiated cohorts (P < 0.001). Increased survival was also associated with significant reductions in lung fibrosis as quantified by CBCT imaging (P < 0.01, 2 weeks post-irradiation). Qualitative histological analyses revealed reduced indications of radiation induced pulmonary injury in animals treated with EV. EV were then subjected to a rigorous proteomic analysis to ascertain the potential bioactive cargo that may prove beneficial in ameliorating radiation-induced normal tissue toxicities in the lung. Proteomics validated several consensus exosome markers (e.g., CD68) and identified major classes of proteins involved in nuclear pore complexes, epigenetics, cell cycle, growth and proliferation, DNA repair, antioxidant function, and cellular metabolism (TCA cycle and oxidative phosphorylation, OXYPHOS). Interestingly, EV were also found to contain mitochondrial components (mtDNA, OXYPHOS protein subunits), which may contribute to the metabolic reprograming and recovery of radiation-injured pulmonary tissue. To evaluate the safety of EV treatments in the context of the radiotherapeutic management of tumors, mice harboring TC1 tumor xenografts were subjected to the same EV treatments shown to forestall lung fibrosis. Data indicated that over the course of one month, no change in the growth of flank tumors between treated and control cohorts was observed. In conclusion, present findings demonstrate that systemic delivery of hESC-derived EV could ameliorate radiation-induced normal tissue complications in the lung, through a variety of potential mechanisms based on EV cargo analysis.

Despite advancements in the radiotherapeutic management of brain malignancies, resultant sequelae include persistent cognitive dysfunction in the majority of survivors. Defining the precise causes of normal tissue toxicity has proven challenging, but the use of preclinical rodent models has suggested that reductions in neurogenesis and microvascular integrity, impaired synaptic plasticity, increased inflammation, and alterations in neuronal structure are contributory if not causal. As such, strategies to reverse these persistent radiotherapy-induced neurological disorders represent an unmet medical need. AM251, a cannabinoid receptor 1 reverse agonist known to facilitate adult neurogenesis and synaptic plasticity, may help to ameliorate radiation-induced CNS impairments. To test this hypothesis, three treatment paradigms were used to evaluate the efficacy of AM251 to ameliorate radiation-induced learning and memory deficits along with disruptions in mood at 4 and 12 weeks postirradiation. Results demonstrated that acute (four weekly injections) and chronic (16 weekly injections) AM251 treatments (1 mg/kg) effectively alleviated cognitive and mood dysfunction in cranially irradiated mice. The beneficial effects of AM251 were exemplified by improved hippocampal- and cortical-dependent memory function on the novel object recognition and object in place tasks, while similar benefits on mood were shown by reductions in depressive- and anxiety-like behaviors on the forced swim test and elevated plus maze. The foregoing neurocognitive benefits were associated with significant increases in newly born (doublecortin+) neurons (1.7-fold), hippocampal neurogenesis (BrdU+/NeuN+mature neurons, 2.5-fold), and reduced expression of the inflammatory mediator HMGB (1.2-fold) in the hippocampus of irradiated mice. Collectively, these findings indicate that AM251 ameliorates the effects of clinically relevant cranial irradiation where overall neurological benefits in memory and mood coincided with increased hippocampal cell proliferation, neurogenesis, and reduced expression of proinflammatory markers.

Cognitive impairment due to cancer and its therapy is a major concern among cancer patients and survivors. Extracellular vesicle (EVs) composition altered by cancer and chemotherapy may affect neurological processes such as neuroplasticity, potentially impacting the cognitive abilities of cancer patients and survivors. We investigated the EV proteome of breast cancer patients with and without cognitive impairment following anthracycline-based chemotherapy from longitudinally collected plasma. EVs were cup-shaped and positive for Flotillin-1 and TSG-101. We identified 517 differentially expressed EV proteins between the cognitive impaired and non-impaired groups during and post-chemotherapy. The observed decreased expression of p2X purinoceptor, cofilin-1, ADAM 10, and dynamin-1 in the plasma EVs of the cognitive impaired group may suggest alterations in the mechanisms underlying synaptic plasticity. The reduced expression of tight junction proteins among cognitive-impaired patients may imply weakening of the blood-brain barrier. These EV protein signatures may serve as a fingerprint that underscores the mechanisms underlying cognitive impairment in cancer patients and survivors.

BackgroundCosmic radiation exposures have been found to elicit cognitive impairments involving a wide-range of underlying neuropathology including elevated oxidative stress, neural stem cell loss, and compromised neuronal architecture. Cognitive impairments have also been associated with sustained microglia activation following low dose exposure to helium ions. Space-relevant charged particles elicit neuroinflammation that persists long-term post-irradiation. Here, we investigated the potential neurocognitive benefits of microglia depletion following low dose whole body exposure to helium ions.MethodsAdult mice were administered a dietary inhibitor (PLX5622) of colony stimulating factor-1 receptor (CSF1R) to deplete microglia 2 weeks after whole body helium irradiation (4He, 30 cGy, 400 MeV/n). Cohorts of mice maintained on a normal and PLX5622 diet were tested for cognitive function using seven independent behavioral tasks, microglial activation, hippocampal neuronal morphology, spine density, and electrophysiology properties 4-6 weeks later.ResultsPLX5622 treatment caused a rapid and near complete elimination of microglia in the brain within 3 days of treatment. Irradiated animals on normal diet exhibited a range of behavioral deficits involving the medial pre-frontal cortex and hippocampus and increased microglial activation. Animals on PLX5622 diet exhibited no radiation-induced cognitive deficits, and expression of resting and activated microglia were almost completely abolished, without any effects on the oligodendrocyte progenitors, throughout the brain. While PLX5622 treatment was found to attenuate radiation-induced increases in post-synaptic density protein 95 (PSD-95) puncta and to preserve mushroom type spine densities, other morphologic features of neurons and electrophysiologic measures of intrinsic excitability were relatively unaffected.ConclusionsOur data suggest that microglia play a critical role in cosmic radiation-induced cognitive deficits in mice and, that approaches targeting microglial function are poised to provide considerable benefit to the brain exposed to charged particles.

Radiation-induced cognitive dysfunction (RICD) is a progressive and debilitating health issue facing patients following cranial radiotherapy to control central nervous system cancers. There has been some success treating RICD in rodents using human neural stem cell (hNSC) transplantation, but the procedure is invasive, requires immunosuppression, and could cause other complications such as teratoma formation. Extracellular vesicles (EV) are nanoscale membrane-bound structures that contain biological contents including mRNA, miRNA, proteins, and lipids that can be readily isolated from conditioned culture media. It has been previously shown that hNSC-derived EV resolves RICD following cranial irradiation using an immunocompromised rodent model. Here, we use immunocompetent wild-type mice to show that hNSC-derived EV treatment administered either intravenously via retro-orbital vein injection or via intracranial transplantation can ameliorate cognitive deficits following 9 Gy head-only irradiation. Cognitive function assessed on the novel place recognition, novel object recognition, and temporal order tasks was not only improved at early (5 weeks) but also at delayed (6 months) postirradiation times with just a single EV treatment. Improved behavioral outcomes were also associated with reduced neuroinflammation as measured by a reduction in activated microglia. To identify the mechanism of action, analysis of EV cargo implicated miRNA (miR-124) as a potential candidate in the mitigation of RICD. Furthermore, viral vector-mediated overexpression of miR-124 in the irradiated brain ameliorated RICD and reduced microglial activation. Our findings demonstrate for the first time that systemic administration of hNSC-derived EV abrogates RICD and neuroinflammation in cranially irradiated wild-type rodents through a mechanism involving miR-124. SIGNIFICANCE: Radiation-induced neurocognitive decrements in immunocompetent mice can be resolved by systemic delivery of hNSC-derived EVs involving a mechanism dependent on expression of miR-124.

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.

Radiotherapy, surgery and the chemotherapeutic agent temozolomide (TMZ) are frontline treatments for glioblastoma multiforme (GBM). However beneficial, GBM treatments nevertheless cause anxiety or depression in nearly 50% of patients. To further understand the basis of these neurological complications, we investigated the effects of combined radiotherapy and TMZ chemotherapy (combined treatment) on neurological impairments using a mouse model. Five weeks after combined treatment, mice displayed anxiety-like behaviors, and at 15 weeks both anxiety- and depression-like behaviors were observed. Relevant to the known roles of the serotonin axis in mood disorders, we found that 5HT1A serotonin receptor levels were decreased by ∼50% in the hippocampus at both early and late time points, and a 37% decrease in serotonin levels was observed at 15 weeks postirradiation. Furthermore, chronic treatment with the selective serotonin reuptake inhibitor fluoxetine was sufficient for reversing combined treatment-induced depression-like behaviors. Combined treatment also elicited a transient early increase in activated microglia in the hippocampus, suggesting therapy-induced neuroinflammation that subsided by 15 weeks. Together, the results of this study suggest that interventions targeting the serotonin axis may help ameliorate certain neurological side effects associated with the clinical management of GBM to improve the overall quality of life for cancer patients.

A decline in cognitive function following cancer treatment is one of the most commonly reported post-treatment symptoms among patients with cancer and those in remission, and include memory, processing speed, and executive function. A clear understanding of cognitive impairment as a result of cancer and its therapy can be obtained by delineating structural and functional changes using brain imaging studies and neurocognitive assessments. There is also a need to determine the underlying mechanisms and pathways that impact the brain and affect cognitive functioning in cancer survivors. Exosomes are small cell-derived vesicles formed by the inward budding of multivesicular bodies, and are released into the extracellular environment via an exocytic pathway. Growing evidence suggests that exosomes contribute to various physiological and pathological conditions, including neurological processes such as synaptic plasticity, neuronal stress response, cell-to-cell communication, and neurogenesis. In this review, we summarize the relationship between exosomes and cancer-related cognitive impairment. Unraveling exosomes' actions and effects on the microenvironment of the brain, which impacts cognitive functioning, is critical for the development of exosome-based therapeutics for cancer-related cognitive impairment.

Cranial radiotherapy, although beneficial for the treatment of brain tumors, inevitably leads to normal tissue damage that can induce unintended neurocognitive complications that are progressive and debilitating. Ionizing radiation exposure has also been shown to compromise the structural integrity of mature neurons throughout the brain, an effect believed to be at least in part responsible for the deterioration of cognitive health. Past work has shown that cranially transplanted human neural stem cells (hNSCs) or their extracellular vesicles (EVs) afforded long-term beneficial effects on many of these cognitive decrements. To provide additional insight into the potential neuroprotective mechanisms of cell-based regenerative strategies, we have analyzed hippocampal neurons for changes in structural integrity and synaptic remodeling after unilateral and bilateral transplantation of hNSCs or EVs derived from those same cells. Interestingly, hNSCs and EVs similarly afforded protection to host neurons, ameliorating the impact of irradiation on dendritic complexity and spine density for neurons present in both the ipsilateral and contralateral hippocampi 1 month following irradiation and transplantation. These morphometric improvements were accompanied by increased levels of glial cell-derived growth factor and significant attenuation of radiation-induced increases in postsynaptic density protein 95 and activated microglia were found ipsi- and contra-lateral to the transplantation sites of the irradiated hippocampus treated with hNSCs or hNSC-derived EVs. These findings document potent far-reaching neuroprotective effects mediated by grafted stem cells or EVs adjacent and distal to the site of transplantation and support their potential as therapeutic agents to counteract the adverse effects of cranial irradiation.

Human stem cell-derived extracellular vesicles (EV) provide many advantages over cell-based therapies for the treatment of functionally compromised tissue beds and organ sites. Here we aimed to highlight multiple administration routes for the potential treatment of various forms of brain injury. Human neural stem cell-derived EV were isolated from conditioned media and administered via three distinct routes: intrahippocampal transplantation, retro-orbital vein injection, and intranasal. EV were administered after which brains were evaluated to determine the capability of EV to translocate into normal tissue. Data showed no significant differences in the amount of EV able to translocate across the brain, indicating the functional equivalence of each administration route to effectively deliver EV to the brain parenchyma. Findings show that both systemic administration routes (retro-orbital vein or intranasal delivery) afforded effective penetrance and perfusion of EV throughout the brain in a minimally invasive manner, and point to a translationally tractable option for treating certain neurological disorders including those resulting from cranial irradiation procedures.

The radiation fields in space define tangible risks to the health of astronauts, and significant work in rodent models has clearly shown a variety of exposure paradigms to compromise central nervous system (CNS) functionality. Despite our current knowledge, sex differences regarding the risks of space radiation exposure on cognitive function remain poorly understood, which is potentially problematic given that 30% of astronauts are women. While work from us and others have demonstrated pronounced cognitive decrements in male mice exposed to charged particle irradiation, here we show that female mice exhibit significant resistance to adverse neurocognitive effects of space radiation. The present findings indicate that male mice exposed to low doses (≤30 cGy) of energetic (400 MeV/n) helium ions (4He) show significantly higher levels of neuroinflammation and more extensive cognitive deficits than females. Twelve weeks following 4He ion exposure, irradiated male mice demonstrated significant deficits in object and place recognition memory accompanied by activation of microglia, marked upregulation of hippocampal Toll-like receptor 4 (TLR4), and increased expression of the pro-inflammatory marker high mobility group box 1 protein (HMGB1). Additionally, we determined that exposure to 4He ions caused a significant decline in the number of dendritic branch points and total dendritic length along with the hippocampus neurons in female mice. Interestingly, only male mice showed a significant decline of dendritic spine density following irradiation. These data indicate that fundamental differences in inflammatory cascades between male and female mice may drive divergent CNS radiation responses that differentially impact the structural plasticity of neurons and neurocognitive outcomes following cosmic radiation exposure.

Human stem cell-derived extracellular vesicles (EV) provide many advantages over cell-based therapies for the treatment of functionally compromised tissue beds and organ sites. Here we sought to determine whether human embryonic stem cell (hESC)-derived EV could resolve in part, the adverse late normal tissue complications associated with exposure of the lung to ionizing radiation. The hESC-derived EV were systemically administered to the mice via the retro-orbital sinus to explore the potential therapeutic benefits following exposure to high thoracic doses of radiation (14 Gy). Data demonstrated that hESC-derived EV treatment significantly improved overall survival of the irradiated cohorts (P < 0.001). Increased survival was also associated with significant reductions in lung fibrosis as quantified by CBCT imaging (P < 0.01, 2 weeks post-irradiation). Qualitative histological analyses revealed reduced indications of radiation induced pulmonary injury in animals treated with EV. EV were then subjected to a rigorous proteomic analysis to ascertain the potential bioactive cargo that may prove beneficial in ameliorating radiation-induced normal tissue toxicities in the lung. Proteomics validated several consensus exosome markers (e.g., CD68) and identified major classes of proteins involved in nuclear pore complexes, epigenetics, cell cycle, growth and proliferation, DNA repair, antioxidant function, and cellular metabolism (TCA cycle and oxidative phosphorylation, OXYPHOS). Interestingly, EV were also found to contain mitochondrial components (mtDNA, OXYPHOS protein subunits), which may contribute to the metabolic reprograming and recovery of radiation-injured pulmonary tissue. To evaluate the safety of EV treatments in the context of the radiotherapeutic management of tumors, mice harboring TC1 tumor xenografts were subjected to the same EV treatments shown to forestall lung fibrosis. Data indicated that over the course of one month, no change in the growth of flank tumors between treated and control cohorts was observed. In conclusion, present findings demonstrate that systemic delivery of hESC-derived EV could ameliorate radiation-induced normal tissue complications in the lung, through a variety of potential mechanisms based on EV cargo analysis.

For decades the field of radiation oncology has sought to improve the therapeutic ratio through innovations in physics, chemistry, and biology. To date, technological advancements in image guided beam delivery techniques have provided clinicians with their best options for improving this critical tool in cancer care. Medical physics has focused on the preferential targeting of tumors while minimizing the collateral dose to the surrounding normal tissues, yielding only incremental progress. However, recent developments involving ultra-high dose rate irradiation termed FLASH radiotherapy (FLASH-RT), that were initiated nearly 50 years ago, have stimulated a renaissance in the field of radiotherapy, long awaiting a breakthrough modality able to enhance therapeutic responses and limit normal tissue injury. Compared to conventional dose rates used clinically (0.1-0.2 Gy/s), FLASH can implement dose rates of electrons or X-rays in excess of 100 Gy/s. The implications of this ultra-fast delivery of dose are significant and need to be re-evaluated to appreciate the fundamental aspects underlying this seemingly unique radiobiology. The capability of FLASH to significantly spare normal tissue complications in multiple animal models, when compared to conventional rates of dose-delivery, while maintaining persistent growth inhibition of select tumor models has generated considerable excitement, as well as skepticism. Based on fundamental principles of radiation physics, radio-chemistry, and tumor vs. normal cell redox metabolism, this article presents a series of testable, biologically relevant hypotheses, which may help rationalize the differential effects of FLASH irradiation observed between normal tissue and tumors.

Response to Ling et al. regarding “An integrated physico-chemical approach for explaining the differential impact of FLASH versus conventional dose rate irradiation on cancer and normal tissue responses”

Ionizing radiation exposure to the brain is common for patients with a variety of CNS related malignancies. This exposure is known to induce structural and functional alterations to the brain, impacting dendritic complexity, spine density and inflammation. Over time, these changes are associated with cognitive decline. However, many of these impacts are only observable long after irradiation. Extracellular vesicles (EVs) are shed from cells in nearly all known tissues, with roles in many disease pathologies. EVs are becoming an important target for identifying circulating biomarkers. The aim of this study is to identify minimally invasive biomarkers of ionizing radiation damage to the CNS that are predictors of late responses that manifest as persistent cognitive impairments. Using a clinically relevant 9 Gy irradiation paradigm, we exposed mice to cranial (head only) irradiation. Using metabolomic and lipidomic profiling, we analyzed their plasma and plasma-derived EVs two days and two weeks post-exposure to detect systemic signs of damage. We identified significant changes associated with inflammation in EVs. Whole-plasma profiling provided further evidence of systemic injury. These studies are the first to demonstrate that profiling of plasma-derived EVs may be used to study clinically relevant markers of ionizing radiation toxicities to the brain.

As NASA prepares for a mission to Mars, concerns regarding the health risks associated with deep space radiation exposure have emerged. Until now, the impacts of such exposures have only been studied in animals after acute exposures, using dose rates ∼1.5×105 higher than those actually encountered in space. Using a new, low dose-rate neutron irradiation facility, we have uncovered that realistic, low dose-rate exposures produce serious neurocognitive complications associated with impaired neurotransmission. Chronic (6 month) low-dose (18 cGy) and dose rate (1 mGy/d) exposures of mice to a mixed field of neutrons and photons result in diminished hippocampal neuronal excitability and disrupted hippocampal and cortical long-term potentiation. Furthermore, mice displayed severe impairments in learning and memory, and the emergence of distress behaviors. Behavioral analyses showed an alarming increase in risk associated with these realistic simulations, revealing for the first time, some unexpected potential problems associated with deep space travel on all levels of neurological function.

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.

PurposeThis review compiles what is known about extracellular vesicles (EVs), their bioactive cargo, and how they might be used to treat radiation-induced brain injury. Radiotherapy (RT) is effective in cancer treatment, but can cause substantial damage to normal central nervous system tissue. Stem cell therapy has been shown to be effective in treating cognitive dysfunction arising from RT, but there remain safety concerns when grafting foreign stem cells into the brain (i.e. immunogenicity, teratoma). These limitations prompted the search for cell-free alternatives, and pointed to EVs that have been shown to have similar ameliorating effects in other tissues and injury models.ConclusionsEVs are nano-scale and lipid-bound vesicles that readily pass the blood-brain barrier. Arguably the most important bioactive cargo within EVs are RNAs, in particular microRNAs (miRNA). A single miRNA can modulate entire gene networks and signalling within the recipient cell. Determining functionally relevant miRNA could lead to therapeutic treatments where synthetically-derived EVs are used as delivery vectors for miRNA. Stem cell-derived EVs can be effective in treating brain injury including radiation-induced cognitive deficits. Of particular interest are systemic modes of administration which obviate the need for invasive procedures.

High-energy charged particles are considered particularly hazardous components of the space radiation environment. Such particles include fully ionized energetic nuclei of helium, silicon, and oxygen, among others. Exposure to charged particles causes reactive oxygen species production, which has been shown to result in neuronal dysfunction and myelin degeneration. Here we demonstrate that mice exposed to high-energy charged particles exhibited alterations in dendritic spine density in the hippocampus, with a significant decrease of thin spines in mice exposed to helium, oxygen, and silicon, compared to sham-irradiated controls. Electron microscopy confirmed these findings and revealed a significant decrease in overall synapse density and in nonperforated synapse density, with helium and silicon exhibiting more detrimental effects than oxygen. Degeneration of myelin was also evident in exposed mice with significant changes in the percentage of myelinated axons and g-ratios. Our data demonstrate that exposure to all types of high-energy charged particles have a detrimental effect, with helium and silicon having more synaptotoxic effects than oxygen. These results have important implications for the integrity of the central nervous system and the cognitive health of astronauts after prolonged periods of space exploration.

It is well known that ionizing radiation-induced toxicity to normal tissue has functional consequences in the brain. However, the underlying molecular alterations have yet to be elucidated. We have previously reported cognitive impairments with concomitant changes in dendritic complexity, spine density and inflammation in mice at 6-24 weeks postirradiation. The goal of this study was to determine whether metabolic changes in the mouse hippocampus after whole-body (4 Gy) or cranial (9 Gy) X-ray irradiation might trigger some of the incipient changes contributing to the persisting pathology in the radiation-injured brain. Metabolomic and lipidomic profiling of hippocampal tissue revealed that radiation induced dyslipidemia in mice at two days and two weeks postirradiation. Strikingly, significant changes were also observed in metabolites of the hexosamine biosynthesis pathway, a finding that was further confirmed using orthogonal methodologies. We hypothesize that these changes in hexosamine metabolism could induce endoplasmic reticulum stress and contribute to radiation-induced cognitive impairments. Taken together, our results show that molecular phenotyping is a valuable approach to identify potentially detrimental pathway perturbations that manifest significantly earlier than gross structural and functional changes in the irradiated brain.

The "FLASH effect" is an increased therapeutic index, that is, reduced normal tissue toxicity for a given degree of anti-cancer efficacy, produced by ultra-rapid irradiation delivered on time scales orders of magnitude shorter than currently conventional in the clinic for the same doses. This phenomenon has been observed in numerous preclinical in vivo tumor and normal tissue models. While the underlying biological mechanism(s) remain to be elucidated, a path to clinical implementation of FLASH can be paved by addressing several critical translational questions. Technological questions pertinent to each beam type (eg , electron, proton, photon) also dictate the logical progression of experimentation required to move forward in safe and decisive clinical trials. Here we review the available preclinical data pertaining to these questions and how they may inform strategies for FLASH cancer therapy clinical trials. [ABSTRACT FROM AUTHOR]

Cognitive dysfunction associated with radiotherapy for cancer treatment has been correlated to several factors, one of which is changes to the dendritic morphology of neuronal cells. Alterations in dendritic geometry and branching patterns are often accompanied by deficits that impact learning and memory. The purpose of this study is to develop a novel predictive model of neuronal dendritic damages caused by exposure to low linear energy transfer (LET) radiation, such as X-rays, γ-rays and high-energy protons. We established in silico representations of mouse hippocampal dentate granule cell layer (GCL) and CA1 pyramidal neurons, which are frequently examined in radiation-induced cognitive decrements. The in silico representations are used in a stochastic model that describes time dependent dendritic damage induced by exposure to low LET radiation. Changes in morphometric parameters, such as total dendritic length, number of branch points and branch number, including the Sholl analysis for single neurons are described by the model. Our model based predictions for different patterns of morphological changes based on energy deposition in dendritic segments (EDDS) will serve as a useful basis to compare specific patterns of morphological alterations caused by EDDS mechanisms.