A second space age spanning omics, platforms and medicine across orbits.

The recent acceleration of commercial, private and multi-national spaceflight has created an unprecedented level of activity in low Earth orbit, concomitant with the largest-ever number of crewed missions entering space and preparations for exploration-class (lasting longer than one year) missions. Such rapid advancement into space from many new companies, countries and space-related entities has enabled a 'second space age'. This era is also poised to leverage, for the first time, modern tools and methods of molecular biology and precision medicine, thus enabling precision aerospace medicine for the crews. The applications of these biomedical technologies and algorithms are diverse, and encompass multi-omic, single-cell and spatial biology tools to investigate human and microbial responses to spaceflight. Additionally, they extend to the development of new imaging techniques, real-time cognitive assessments, physiological monitoring and personalized risk profiles tailored for astronauts. Furthermore, these technologies enable advancements in pharmacogenomics, as well as the identification of novel spaceflight biomarkers and the development of corresponding countermeasures. In this Perspective, we highlight some of the recent biomedical research from the National Aeronautics and Space Administration, Japan Aerospace Exploration Agency, European Space Agency and other space agencies, and detail the entrance of the commercial spaceflight sector (including SpaceX, Blue Origin, Axiom and Sierra Space) into aerospace medicine and space biology, the first aerospace medicine biobank, and various upcoming missions that will utilize these tools to ensure a permanent human presence beyond low Earth orbit, venturing out to other planets and moons.

Galactic cosmic radiation exposure causes multifaceted neurocognitive impairments.

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 (< 0.3 mm) and larger (> 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.

Long-term neurocognitive benefits of FLASH radiotherapy driven by reduced reactive oxygen species.

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.

Detrimental impacts of mixed-ion radiation on nervous system function.

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.

Chronic Low Dose Neutron Exposure Results in Altered Neurotransmission Properties of the Hippocampus-Prefrontal Cortex Axis in Both Mice and Rats.

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.

Acute, Low-Dose Neutron Exposures Adversely Impact Central Nervous System Function.

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.

Mitigation of helium irradiation-induced brain injury by microglia depletion.

BACKGROUND: Cosmic 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. METHODS: Adult 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. RESULTS: PLX5622 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. CONCLUSIONS: Our 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.

Role of Exosomes in Cancer-Related Cognitive Impairment.

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 grafting of stem cell-derived microvesicles improves cognition and reduces neuropathology in the irradiated brain.

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 head-only 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.

Multiple Forms of Endocannabinoid and Endovanilloid Signaling Regulate the Tonic Control of GABA Release.

Persistent CB1 cannabinoid receptor activity limits neurotransmitter release at various synapses throughout the brain. However, it is not fully understood how constitutively active CB1 receptors, tonic endocannabinoid signaling, and its regulation by multiple serine hydrolases contribute to the synapse-specific calibration of neurotransmitter release probability. To address this question at perisomatic and dendritic GABAergic synapses in the mouse hippocampus, we used a combination of paired whole-cell patch-clamp recording, liquid chromatography/tandem mass spectrometry, stochastic optical reconstruction microscopy super-resolution imaging, and immunogold electron microscopy. Unexpectedly, application of the CB1 antagonist and inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], but not the neutral antagonist NESS0327 [8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-5,6-dihydro-4H-benzo[2,3]cyclohepta[2,4-b]pyrazole-3-carboxamine], significantly increased synaptic transmission between CB1-positive perisomatic interneurons and CA1 pyramidal neurons. JZL184 (4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate), a selective inhibitor of monoacylglycerol lipase (MGL), the presynaptic degrading enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), elicited a robust increase in 2-AG levels and concomitantly decreased GABAergic transmission. In contrast, inhibition of fatty acid amide hydrolase (FAAH) by PF3845 (N-pyridin-3-yl-4-[[3-[5-(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide) elevated endocannabinoid/endovanilloid anandamide levels but did not change GABAergic synaptic activity. However, FAAH inhibitors attenuated tonic 2-AG increase and also decreased its synaptic effects. This antagonistic interaction required the activation of the transient receptor potential vanilloid receptor TRPV1, which was concentrated on postsynaptic intracellular membrane cisternae at perisomatic GABAergic symmetrical synapses. Interestingly, neither AM251, JZL184, nor PF3845 affected CB1-positive dendritic interneuron synapses. Together, these findings are consistent with the possibility that constitutively active CB1 receptors substantially influence perisomatic GABA release probability and indicate that the synaptic effects of tonic 2-AG release are tightly controlled by presynaptic MGL activity and also by postsynaptic endovanilloid signaling and FAAH activity. SIGNIFICANCE STATEMENT: Tonic cannabinoid signaling plays a critical role in the regulation of synaptic transmission. However, the mechanistic details of how persistent CB1 cannabinoid receptor activity inhibits neurotransmitter release have remained elusive. Therefore, electrophysiological recordings, lipid measurements, and super-resolution imaging were combined to elucidate those signaling molecules and mechanisms that underlie tonic cannabinoid signaling. The findings indicate that constitutive CB1 activity has pivotal function in the tonic control of hippocampal GABA release. Moreover, the endocannabinoid 2-arachidonoylglycerol (2-AG) is continuously generated postsynaptically, but its synaptic effect is regulated strictly by presynaptic monoacylglycerol lipase activity. Finally, anandamide signaling antagonizes tonic 2-AG signaling via activation of postsynaptic transient receptor potential vanilloid TRPV1 receptors. This unexpected mechanistic diversity may be necessary to fine-tune GABA release probability under various physiological and pathophysiological conditions.

Irradiation of Neurons with High-Energy Charged Particles: An In Silico Modeling Approach.

In this work, a stochastic computational model of microscopic energy deposition events is used to study for the first time damage to irradiated neuronal cells of the mouse hippocampus. An extensive library of radiation tracks for different particle types is created to score energy deposition in small voxels and volume segments describing a neuron's morphology that later are sampled for given particle fluence or dose. Methods included the construction of in silico mouse hippocampal granule cells from neuromorpho.org with spine and filopodia segments stochastically distributed along the dendritic branches. The model is tested with high-energy (56)Fe, (12)C, and (1)H particles and electrons. Results indicate that the tree-like structure of the neuronal morphology and the microscopic dose deposition of distinct particles may lead to different outcomes when cellular injury is assessed, leading to differences in structural damage for the same absorbed dose. The significance of the microscopic dose in neuron components is to introduce specific local and global modes of cellular injury that likely contribute to spine, filopodia, and dendrite pruning, impacting cognition and possibly the collapse of the neuron. Results show that the heterogeneity of heavy particle tracks at low doses, compared to the more uniform dose distribution of electrons, juxtaposed with neuron morphology make it necessary to model the spatial dose painting for specific neuronal components. Going forward, this work can directly support the development of biophysical models of the modifications of spine and dendritic morphology observed after low dose charged particle irradiation by providing accurate descriptions of the underlying physical insults to complex neuron structures at the nano-meter scale.

Cranial irradiation compromises neuronal architecture in the hippocampus.

Cranial irradiation is used routinely for the treatment of nearly all brain tumors, but may lead to progressive and debilitating impairments of cognitive function. Changes in synaptic plasticity underlie many neurodegenerative conditions that correlate to specific structural alterations in neurons that are believed to be morphologic determinants of learning and memory. To determine whether changes in dendritic architecture might underlie the neurocognitive sequelae found after irradiation, we investigated the impact of cranial irradiation (1 and 10 Gy) on a range of micromorphometric parameters in mice 10 and 30 d following exposure. Our data revealed significant reductions in dendritic complexity, where dendritic branching, length, and area were routinely reduced (>50%) in a dose-dependent manner. At these same doses and times we found significant reductions in the number (20-35%) and density (40-70%) of dendritic spines on hippocampal neurons of the dentate gyrus. Interestingly, immature filopodia showed the greatest sensitivity to irradiation compared with more mature spine morphologies, with reductions of 43% and 73% found 30 d after 1 and 10 Gy, respectively. Analysis of granule-cell neurons spanning the subfields of the dentate gyrus revealed significant reductions in synaptophysin expression at presynaptic sites in the dentate hilus, and significant increases in postsynaptic density protein (PSD-95) were found along dendrites in the granule cell and molecular layers. These findings are unique in demonstrating dose-responsive changes in dendritic complexity, synaptic protein levels, spine density and morphology, alterations induced in hippocampal neurons by irradiation that persist for at least 1 mo, and that resemble similar types of changes found in many neurodegenerative conditions.

Radiomics approach for identifying radiation-induced normal tissue toxicity in the lung.

Radiomic features were used in efforts to characterize radiation-induced normal tissue injury as well as identify if human embryonic stem cell (hESC) derived Extracellular Vesicle (EV) treatment could resolve certain adverse complications. A cohort of mice (n=12/group) were given whole lung irradiation (3×8Gy), local irradiation to the right lung apex (3×12Gy), or no irradiation. The hESC-derived EVs were systemically administered three times via retro-orbital injection immediately after each irradiation. Cone-Beam Computed Tomography (CBCT) images were acquired at baseline and 2 weeks after the final radiation/EV treatment. Whole lung image segmentation was performed and radiomic features were extracted with wavelet filtering applied. A total of 851 features were extracted per image and recursive feature elimination was used to refine, train and validate a series of random forest classification models. Classification models trained to identify irradiated from unirradiated animals or EV treated from vehicle-injected animals achieved high prediction accuracies (94% and 85%). In addition, radiomic features from the locally irradiated dataset showed significant radiation impact and EV sparing effects that were absent in the unirradiated left lung. Our data demonstrates that radiomics has the potential to characterize radiation-induced lung injury and identify therapeutic efficacy at early timepoints.

Radiomics approach for identifying radiation-induced normal tissue toxicity in the lung.

The rapidly evolving field of radiomics has shown that radiomic features are able to capture characteristics of both tumor and normal tissue that can be used to make accurate and clinically relevant predictions. In the present study we sought to determine if radiomic features can characterize the adverse effects caused by normal tissue injury as well as identify if human embryonic stem cell (hESC) derived extracellular vesicle (EV) treatment can resolve certain adverse complications. A cohort of 72 mice (n = 12 per treatment group) were exposed to X-ray radiation to the whole lung (3 × 8 Gy) or to the apex of the right lung (3 × 12 Gy), immediately followed by retro-orbital injection of EVs. Cone-Beam Computed Tomography images were acquired before and 2 weeks after treatment. In total, 851 radiomic features were extracted from the whole lungs and < 20 features were selected to train and validate a series of random forest classification models trained to predict radiation status, EV status and treatment group. It was found that all three classification models achieved significantly high prediction accuracies on a validation subset of the dataset (AUCs of 0.91, 0.86 and 0.80 respectively). In the locally irradiated lung, a significant difference between irradiated and unirradiated groups as well as an EV sparing effect were observed in several radiomic features that were not seen in the unirradiated lung (including wavelet-LLH Kurtosis, wavelet HLL Large Area High Gray Level Emphasis, and Gray Level Non-Uniformity). Additionally, a radiation difference was not observed in a secondary comparison cohort, but there was no impact of imaging machine parameters on the radiomic signature of unirradiated mice. Our data demonstrate that radiomics has the potential to identify radiation-induced lung injury and could be applied to predict therapeutic efficacy at early timepoints.

Extracellular vesicles from GABAergic but not glutamatergic neurons protect against neurological dysfunction following cranial irradiation.

Cranial irradiation used to control brain malignancies invariably leads to progressive and debilitating declines in cognition. Clinical efforts implementing hippocampal avoidance and NMDAR antagonism, have sought to minimize dose to radiosensitive neurogenic regions while normalizing excitatory/inhibitory (E/I) tone. Results of these trials have yielded only marginal benefits to cognition, prompting current studies to evaluate the potential of systemic extracellular vesicle (EV) therapy to restore neurocognitive functionality in the irradiated brain. Here we tested the hypothesis that EVs derived from inhibitory but not excitatory neuronal cultures would prove beneficial to cognition and associated pathology. Rats subjected to a clinically relevant, fractionated cranial irradiation paradigm were given multiple injections of either GABAergic- or glutamatergic-derived EV and subjected to behavioral testing. Rats treated with GABAergic but not glutamatergic EVs showed significant improvements on hippocampal- and cortical-dependent behavioral tasks. While each treatment enhanced levels of the neurotrophic factors BDNF and GDNF, only GABAergic EVs preserved granule cell neuron dendritic spine density. Additional studies conducted with GABAergic EVs, confirmed significant benefits on amygdala-dependent behavior and modest changes in synaptic plasticity as measured by long-term potentiation. These data point to a potentially more efficacious approach for resolving radiation-induced neurological deficits, possibly through a mechanism able to restore homeostatic E/I balance.

Targeting dysfunctional endocannabinoid signaling in a mouse model of Gulf War illness.

Gulf War Illness (GWI) is a chronic disorder characterized by a heterogeneous set of symptoms that include pain, fatigue, anxiety, and cognitive impairment. These are thought to stem from damage caused by exposure under unpredictable stress to toxic Gulf War (GW) chemicals, which include pesticides, nerve agents, and prophylactic drugs. We hypothesized that GWI pathogenesis might be rooted in long-lasting disruption of the endocannabinoid (ECB) system, a signaling complex that serves important protective functions in the brain. Using a mouse model of GWI, we found that tissue levels of the ECB messenger, anandamide, were significantly reduced in the brain of diseased mice, compared to healthy controls. In addition, transcription of the Faah gene, which encodes for fatty acid amide hydrolase (FAAH), the enzyme that deactivates anandamide, was significant elevated in prefrontal cortex of GWI mice and brain microglia. Behavioral deficits exhibited by these animals, including heightened anxiety-like and depression-like behaviors, and defective extinction of fearful memories, were corrected by administration of the FAAH inhibitor, URB597, which normalized brain anandamide levels. Furthermore, GWI mice displayed unexpected changes in the microglial transcriptome, implying persistent dampening of homeostatic surveillance genes and abnormal expression of pro-inflammatory genes upon immune stimulation. Together, these results suggest that exposure to GW chemicals produce a deficit in brain ECB signaling which is associated with persistent alterations in microglial function. Pharmacological normalization of anandamide-mediated ECB signaling may offer an effective therapeutic strategy for ameliorating GWI symptomology.

Structural plasticity of pyramidal cell neurons measured after FLASH and conventional dose-rate irradiation.

Evidence shows that ultra-high dose-rate FLASH-radiotherapy (FLASH-RT) protects against normal tissue complications and functional decrements in the irradiated brain. Past work has shown that radiation-induced cognitive impairment, neuroinflammation and reduced structural complexity of granule cell neurons were not observed to the same extent after FLASH-RT (> MGy/s) compared to conventional dose-rate (CONV, 0.1 Gy/s) delivery. To explore the sensitivity of different neuronal populations to cranial irradiation and dose-rate modulation, hippocampal CA1 and medial prefrontal cortex (PFC) pyramidal neurons were analyzed by electron and confocal microscopy. Neuron ultrastructural analyses by electron microscopy after 10 Gy FLASH- or CONV-RT exposures indicated that irradiation had little impact on dendritic complexity and synapse density in the CA1, but did increase length and head diameter of smaller non-perforated synapses. Similarly, irradiation caused no change in PFC prelimbic/infralimbic axospinous synapse density, but reductions in non-perforated synapse diameters. While irradiation resulted in thinner myelin sheaths compared to controls, none of these metrics were dose-rate sensitive. Analysis of fluorescently labeled CA1 neurons revealed no radiation-induced or dose-rate-dependent changes in overall dendritic complexity or spine density, in contrast to our past analysis of granule cell neurons. Super-resolution confocal microscopy following a clinical dosing paradigm (3×10Gy) showed significant reductions in excitatory vesicular glutamate transporter 1 and inhibitory vesicular GABA transporter puncta density within the CA1 that were largely dose-rate independent. Collectively, these data reveal that, compared to granule cell neurons, CA1 and mPFC neurons are more radioresistant irrespective of radiation dose-rate.

Acute Hypoxia Does Not Alter Tumor Sensitivity to FLASH Radiation Therapy.

PURPOSE: Tumor hypoxia is a major cause of treatment resistance, especially to radiation therapy at conventional dose rate (CONV), and we wanted to assess whether hypoxia does alter tumor sensitivity to FLASH. METHODS AND MATERIALS: We engrafted several tumor types (glioblastoma [GBM], head and neck cancer, and lung adenocarcinoma) subcutaneously in mice to provide a reliable and rigorous way to modulate oxygen supply via vascular clamping or carbogen breathing. We irradiated tumors using a single 20-Gy fraction at either CONV or FLASH, measured oxygen tension, monitored tumor growth, and sampled tumors for bulk RNAseq and pimonidazole analysis. Next, we inhibited glycolysis with trametinib in GBM tumors to enhance FLASH efficacy. RESULTS: Using various subcutaneous tumor models, and in contrast to CONV, FLASH retained antitumor efficacy under acute hypoxia. These findings show that in addition to normal tissue sparing, FLASH could overcome hypoxia-mediated tumor resistance. Follow-up molecular analysis using RNAseq profiling uncovered a FLASH-specific profile in human GBM that involved cell-cycle arrest, decreased ribosomal biogenesis, and a switch from oxidative phosphorylation to glycolysis. Glycolysis inhibition by trametinib enhanced FLASH efficacy in both normal and clamped conditions. CONCLUSIONS: These data provide new and specific insights showing the efficacy of FLASH in a radiation-resistant context, proving an additional benefit of FLASH over CONV.

Antitumor Effect by Either FLASH or Conventional Dose Rate Irradiation Involves Equivalent Immune Responses.

PURPOSE: The capability of ultrahigh dose rate FLASH radiation therapy to generate the FLASH effect has opened the possibility to enhance the therapeutic index of radiation therapy. The contribution of the immune response has frequently been hypothesized to account for a certain fraction of the antitumor efficacy and tumor kill of FLASH but has yet to be rigorously evaluated. METHODS AND MATERIALS: To investigate the immune response as a potentially important mechanism of the antitumor effect of FLASH, various murine tumor models were grafted either subcutaneously or orthotopically into immunocompetent mice or in moderately and severely immunocompromised mice. Mice were locally irradiated with single dose (20 Gy) or hypofractionated regimens (3 × 8 or 2 × 6 Gy) using FLASH (≥2000 Gy/s) and conventional (CONV) dose rates (0.1 Gy/s), with/without anti-CTLA-4. Tumor growth was monitored over time and immune profiling performed. RESULTS: FLASH and CONV 20 Gy were isoeffective in delaying tumor growth in immunocompetent and moderately immunodeficient hosts and increased tumor doubling time to >14 days versus >7 days in control animals. Similar observations were obtained with a hypofractionated scheme, regardless of the microenvironment (subcutaneous flank vs ortho lungs). Interestingly, in profoundly immunocompromised mice, 20 Gy FLASH retained antitumor activity and significantly increased tumor doubling time to >14 days versus >8 days in control animals, suggesting a possible antitumor mechanism independent of the immune response. Analysis of the tumor microenvironment showed similar immune profiles after both irradiation modalities with significant decrease of lymphoid cells by ∼40% and a corresponding increase of myeloid cells. In addition, FLASH and CONV did not increase transforming growth factor-ß1 levels in tumors compared with unirradiated control animals. Furthermore, when a complete and long-lasting antitumor response was obtained (>140 days), both modalities of irradiation were able to generate a long-term immunologic memory response. CONCLUSIONS: The present results clearly document that the tumor responses across multiple immunocompetent and immunodeficient mouse models are largely dose rate independent and simultaneously contradict a major role of the immune response in the antitumor efficacy of FLASH. Therefore, our study indicates that FLASH is as potent as CONV in modulating antitumor immune response and can be used as an immunomodulatory agent.

Navigating the Critical Translational Questions for Implementing FLASH in the Clinic.

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.