Task Progress:
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Protons
To date, two proton irradiation campaigns have been conducted and provided biological replicates for measurements. Subsequent experiments examine simulated galactic cosmic radiation (GCR) spectra for which protons are the main component. A battery of 15 behavioral tests assesses cognitive, affective, and sensorimotor performance in both male and female mice at 1-2, 3-5, & 9 months post irradiation/IR (referred to as “1 month,” “3 month,” and “9 month” time points) 9-month measurements were not originally planned but resulted from Covid-19 related restrictions on animal care facility access and have been continued. Briefly, in cases where 50 cGy proton exposure resulted in altered behavioral parameters, the percent change from sham values ranged up to 74% and the magnitude of change from fractionated exposures often exceeded that for acute exposures. This included: Y-maze, elevated plus maze, light-dark box, open field exploration, novel object and novel place recognition, sociability and social recognition, modified balance beam, hindlimb unloading, and forced swim tests. Fractionated exposures were notably more effective for step-through passive avoidance. Other behaviors were not significantly affected by irradiation. To assess neuronal network function, expression of the immediate early gene c-fos driven by fear memory (passive avoidance) was measured in selected brain regions of male mice at 3 months post IR. Passive avoidance testing elicited strong gene expression in multiple brain regions compared to home cage controls. Prior radiation exposure resulted in altered c-fos expression reflecting changes in behaviorally driven network activity.
To determine which molecular markers and signaling pathways underlie outcomes of the treatment conditions we used NanoString® technology to profile mRNA expression at 3 months post irradiation of male mice in orbitofrontal cortex, hippocampus, and cerebellum. A set of 770 genes from the nCounter® Mouse Neuropathology panel for 23 fundamental pathways were examined and were characterized according to annotations for: structural integrity, metabolism, neuroinflammation, neuron-glia interaction, plasticity and aging, and neurotransmission. Expression patterns varied with radiation dose rate and brain region. In hippocampus, pathway activation was generally similar for fractionated vs acute expression while in frontal cortex fractionated exposure differed substantially from either sham or acute exposures. When the data was analyzed for the top genes based on absolute fold expression value, the 8 highest differentially expressed genes were associated with oxidative stress, protein homeostasis, and inflammation. They included: Arc and Fos which are immediate early genes expressed after synaptic activity and oxidative stress, Cp (Ceruloplasmin) which may have antioxidant activity in astrocytes, regulate monoamine pathways and serve as a copper transporter, Des (Desmin) which is associated with astrocyte activation and is expressed along with GFAP, Nqo1 (NADPH dehydrogenase quinone) which is associated with adaptation to stress and may regulate Poly (ADP-ribose) formation (DNA repair, apoptosis) and proteasome activity on denatured proteins, Pla2g4 (Phospholipase A2) which regulates signaling in neuroinflammation and oxidative stress, Psmb9 (Proteosome subunit 9) which regulates protein degradation, and Shh (Sonic Hedgehog) which is a central nervous system (CNS) morphogen that also regulates autophagy and shows protective activity for neurogenesis and oxidative stress. Similar differentially expressed gene sets were observed for male and female mice exposed to simulated galactic cosmic rays but there were significant sex differences in both hippocampus and frontal cortex.
Chronic Mild Stress
During the Covid-19 driven delay in the use of (Brookhaven National Laboratory) BNL irradiation facilities we conducted experiments using the chronic mild stress (CMS) model to simulate combined stressors experienced during spaceflight. This well-vetted model delivered mild stress from disruption of cage environment, lighting, social interactions, predator cues, etc., over a 4-week period as a surrogate for the multisensory set of non-radiation space flight stressors. The CMS exposures were conducted from 1/4/2021 to 2/1/2021 and tests were performed at 1-2 or 4-5 weeks after the exposure. We measured selected behavioral outcome measures, as described above, along with stress hormone, corticosterone, and a suite of cytokine. Several anxiety-related outcome measures showed strong increases at 1- and 4-weeks post CMS including light-dark, open field, and elevated plus maze tests while cognitive measures were less responsive. Corticosterone levels were elevated as expected. In the future the CMS regimen will be combined with a 0.5 Gy proton exposure to characterize interactions of the combined stresses.
GCRsim
236 male mice were exposed to 50 cGy GCRsim (sham, acute, and fractionated regimens) and 2 Gy fractionated gamma rays during the NASA Space Radiation Laboratory (NSRL) 2021 campaign in April/May 2021 by special arrangement between NASA and BNL. Behavioral batteries described above for protons were conducted at 1-, 3- and 9-month time points with final data acquisition in March 2022. 3-month c-fos expression histological samples have been archived and are undergoing counting. Brain tissue samples from 7 regions were frozen for biochemical analysis and hemibrains fixed for histology. 176 female and 60 male mice were exposed to GCRsim and gamma rays from 4/11/22 to 5/6/22 during the NSRL22A campaign and shipped back to Loma Linda University where they were quarantined for 7 weeks after which behavioral testing began. Behavioral testing was completed in November 2022 and tissues were archived for histology and biochemistry. Fixed samples were processed for c-fos and neuroinflammatory marker immunohistochemistry and imaging and quantification is still in progress. To better understand the shape of the dose-response curve and to improve RBE estimates, a final cohort of 180 female (120) and male (60) mice were irradiated acutely during the NSRL23A/B campaign (April 25 and 26, 2023) with the 33-ion GCRsim field at doses of 0, 15, 25, 50 and 75 cGy complemented by a 2 Gy acute exposure to 137-Cs gamma rays. These were processed for behavioral and histological endpoints as per previous animals exposed to fractionated GCRsim.
GCRsim exposure elicited a number of behavioral changes in male mice at all three time points, which also allowed us to follow the time course for certain outcome measures. Preliminary results indicate that GCRsim exposure elicited changes in many outcome measures, and gamma rays were also effective, which enabled estimation of (relative biological effectiveness) RBE values. GCRsim exposures did not significantly affect distance or time-in-location measures in the open field, while gamma rays increased locomotion and reduced freezing. Light-dark box tests revealed increased locomotion and reduced anxiety (increased time in light zone and transitions), which was time course dependent. Balance beam revealed elevated locomotion and reduced anxiety and hindlimb unloading depression-like behavior (learned helplessness) revealed enhancement of “depression” at 1 month which resolved at 3 and 9 months. Working memory (Y maze spontaneous alternation) showed enhancement at late times with fractionated GCRsim and gamma rays. Fear memory (passive avoidance) was insensitive to GCRsim. For females subjected to the same behavioral battery, there was generally a smaller effect than for males. Open field distance and center time measures were reduced in fractionated exposure animals but time immobile was increased. Novel object recognition memory was not significantly affected. Elevated plus maze measures indicated reduced anxiety in males but not females while females were found to move 25% more than males. Y maze working memory and passive avoidance fear memory measures were not significantly altered, nor was depression-like behavior in the hindlimb unloading test. For the dose-response series of acute exposures in females, there were similar outcomes to those described above but the magnitude of changes was subtle. Both linear and non-linear dose responses were observed depending on endpoint measures.
We are exploring the use of data transformed to Z-scores and Hedge’s g or Cohen’s d effect sizes to clarify which outcome measures are the most robust, not just the most significant based on sampling statistics. To reduce the influence of particular experimental set-ups we are combining Z-scores for selected parameters into integrated Z-scores for broadly defined behavioral domains such as emotionality and locomotory behaviors. For example, locomotory behavior is assessed in open field, Y-maze, novel object, and elevated plus maze arenas, each of which has its own geometry, lighting, and external cues. Distance, entry, and speed parameters from these arenas all reflect locomotory behavior so that Z-scores at the individual animal level for the multiple parameters can be combined and normalized to a consensus score to eliminate noise and test bias. Dose Rate Effectiveness Factor (DREF) and RBE values calculated from these consensus scores are expected to be superior to those from individual test parameters.
Histological samples from NSRL23A/B mice are being photographed for quantitation of IBA-1 and GFAP glial activation markers reflecting neuroinflammation. mRNA samples from NSRL22 and 21 mice were prepared for transcriptomics analysis using the NanoString® technology reported for protons. The data have been reported back from the vendor and are undergoing analysis for pathway scores and differential expression. A first look suggests that gene expression patterns are dependent on sex, brain region, and radiation type. Despite the fact that protons numerically, and as a dose fraction, dominate the GCRsim field, the GCRsim expression patterns differ significantly from those observed for protons.
DREF Estimates
Characterization of dose rate effects by a simple parameter like DREF is problematic for CNS outcome measures. Behavioral outcome measures reflect complex interactions of motivation, sensory and motor function, emotional status, etc., and outcome parameter values reflect the balance between conflicting behavioral drives such as anxiety and curiosity. Thus, deviations from the control values can be positive or negative – reflecting an altered but stable new state not necessarily interpretable as detrimental – and the raw measures come in a variety of units such as distance, time, force, etc., making them hard to pool. We have turned to standardized effect size measures such as Cohen’s d, Hedge’s g, and Z-scores to transform the data to a single metric (units of standard deviation from control means) which has enabled us to develop distributions of DREF values for proton and GCRsim exposures. Preliminary DREFs for all pooled behavioral measures were approximately 1.65 for both protons and GCRsim, similar to the estimate of 2 used by radiation risk advisory bodies such as the International Commission on Radiological Protection (ICRP). Male and female values were similar. RBE estimates were also derived from effect size measures with linear interpolation of dose responses and yielded values of approximately 3.9. These values were limited to behavioral data sets which were normally distributed and for which effect sizes (Hedge’s g) exceeded 0.2. Effect size and integrated Z-score approaches will be used for DREF and RBE estimates and the dose-response curve from the NSRL23A/B experiments will be used to correct for curvature in the GCRsim data which currently assumes linearity.
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Articles in Peer-reviewed Journals
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Koenig-Zanoff M, Frattini V, Nimmagadda H, Feng X, Jones T, Nelson G, Ferguson AR, Rosi S. "The impact of deep space radiation on cognitive performance: From biological sex to biomarkers to countermeasures. " Sci Adv. 2021 Oct 15;7(42):eabg6702. https://doi.org/10.1186/s12916-022-02705-6 ; PMID: 34652936; PMCID: PMC8519563 , Oct-2021
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Papers from Meeting Proceedings
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Nelson G, Jones, T, Stanbouly, S, Hartman, R, Grue K, and S Rosi. "Dose rate effects of space radiation on the mouse nervous system." 2022 NASA Human Research Program Investigators’ Workshop. 2022 NASA Human Research Program Investigators’ Workshop. Presentation. Abstract 11334-000270. , Feb-2022
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Papers from Meeting Proceedings
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Nelson G, Jones T, Stanbouly S, Grue K, Rosi S. "Responses of the Central Nervous System to Simulated Cosmic Rays: Unique or Not?" 2022 NASA Human Research Program Investigators’ Workshop. 2022 NASA Human Research Program Investigators’ Workshop. Presentation. Abstract 11334-000547. , Feb-2022
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Papers from Meeting Proceedings
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Nelson G, Jones T, Stanbouly S, Grue K, Rosi S. "Charged Particle Radiation Dose Rate Effectiveness Factors for Mouse CNS." 68th Annual Meeting of Radiation Research Society, Big Island, HI, October 16-19, 2022. 68th Annual Meeting of Radiation Research Society, Big Island, HI, October 16-19, 2022. Poster PS3-06. , Oct-2022
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Papers from Meeting Proceedings
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Nelson GA, Jones T, Stanbouly S. "Dose Rate Effects of Space Radiation on the Mouse Nervous System." 2023 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 7-9, 2023. 2023 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 7-9, 2023. Presentation. DS24-1. , Feb-2023
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Papers from Meeting Proceedings
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Nelson G, Jones T, Stanbouly S. "Responses of the mouse nervous system to simulated cosmic ray exposure and effects of dose rate." 17th International Congress on Radiation Research. Montreal, Canada, August 27-30, 2023. 17th International Congress on Radiation Research. Montreal, Canada, August 27-30, 2023. Presentation. Symposium talk S12-04. , Aug-2023
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Papers from Meeting Proceedings
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Saha JP, Nelson G, Sishc B, Zawaski J, Elgart SR. "Conclusions of a mini technical interchange meeting on mechanisms and pathways common between adverse health outcomes from exposures to space radiation." 17th International Congress on Radiation Research. Montreal, Canada, August 27-30, 2023. 17th International Congress on Radiation Research. Montreal, Canada, August 27-30, 2023. Poster PS3-54. , Aug-2023
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Papers from Meeting Proceedings
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Sishc BJ, Nelson G. "Biological Space Radiation Countermeasures to Enable Long Duration Exploration Missions." 17th International Congress on Radiation Research. Montreal, Canada, August 27-30, 2023. 17th International Congress on Radiation Research. Montreal, Canada, August 27-30, 2023. Poster PS3-61. , Aug-2023
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Papers from Meeting Proceedings
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Nelson G, Jones T, Stanbouly S. "Effects of Radiation Dose Rate on the Mouse Nervous System." 2024 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 13-16, 2024. 2024 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 13-16, 2024. Presentation 1645630. , Feb-2024
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Papers from Meeting Proceedings
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Reinsch S, Elgart SR, Guida P, Nelson G, Saha J, Santa Maria S, Sishc B, Weeks J, Zawaski J. "NASA Space Health Impacts for the NASA Experience (SHINE) Training Program: Space Radiation Curriculum." 2024 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 13-16, 2024. 2024 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 13-16, 2024. Presentation 1645749. , Feb-2024
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Papers from Meeting Proceedings
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Alwood J, Antonsen E, Dev S, Nelson G, Reynolds R, Shahid A. "DAG Studies for the Behavioral Medicine Risk." 2024 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 13-16, 2024. 2024 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 13-16, 2024. Poster 1650429. , Feb-2024
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