Task Progress:
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To date, two proton irradiation campaigns have been conducted and provided biological replicates for measurements. Future experiments will examine simulated GCR spectra for which protons are the main component. A battery of 15 behavioral tests assessed cognitive, affective, and sensorimotor performance in both male (1, 3, & 9 months post IR) and female mice (3 months post IR). 9-month measurements in males were not originally planned but resulted from Covid-19 related restrictions on animal care facility access. In cases where 0.5 Gy 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, tail suspension, and forced swim tests. Fractionated exposures were notably more effective for step-through passive avoidance. Defensive marble burying, grip strength, water maze, and accelerating rotarod coordination behaviors were not significantly affected by irradiation and animals maintained good learning ability in training phases.
To assess neuronal network function, expression of the immediate early gene c-fos was measured in selected brain regions of male mice at 3 months post IR. 90 min after completion of the 24-hour recall phase of passive avoidance testing mice were sacrificed and brain tissue prepared for counts of cells expressing c-fos. Regions of interest (ROI) used for cell counting included: orbitofrontal cortex, cingulate cortex, hippocampus, caudate putamen, thalamic and hypothalamic nuclei, amygdala, and cerebellum. Passive avoidance testing elicited strong gene expression in all of these regions compared to home cage controls. Prior radiation exposure resulted in altered c-fos expression reflecting changes in behaviorally driven network activity. Percent changes in c-fos(+) cell number in ROIs of irradiated animals compared to shams were typically of the order 50% and the ratios of change (acute vs fractionated treatment samples) were typically 1.1 to 1.8. Three regions showed notable differences in expression levels as a function of treatment: hippocampus CA1 & CA3 fields and cerebellum granular layers. c-fos imaging is currently in progress for a second behavioral “task,” tail suspension, which is expected to drive neuronal pathways associated with anxiety and depression-like behaviors.
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. Five replicate brain regions were used for each treatment condition (sham, acute, fractionated). A set of 760 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 & 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. Pathways involving activated microglia, growth factors and trophins, nerve structure as well as endothelial cells were the most affected by dose rate and region.
During the Covid-19 driven delay in the use of BNL irradiation facilities we have initiated exploratory experiments using the chronic mild stress (CMS) model to simulate combined stressors experienced during space flight. This well-vetted model will deliver mild stress over a 4-week period to simulate the set of non-radiation space flight stressors and measure selected outcome measures as described above. Then the CMS regimen will be combined with a 0.5 Gy proton exposure to characterize interactions of the combined stresses.
Characterization of the dose rate effects by a simple parameter like DREF is problematic for CNS outcome measures which are deterministic and exhibit non-linear dose responses. 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. Non-DREF methods for comparing dose rate effects will be considered as the study progresses.
To date, presentations of experimental finding have been made to the 2019 and 2020 annual meetings of the Radiation Research Society and to the 2020 NASA Human Research Program (HRP) Investigators’ Working Group. Publications on proton-irradiations conducted in 2019 – 2020 are in preparation.
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Abstracts for Journals and Proceedings
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Nelson G, Jones T, Stanbouly S, Tolan B, Wroe A, Hartman R. "Effects of Dose Rate on Responses of the Brain to Charged Particles." 65th Annual Meeting of the Radiation Research Society, San Diego, CA, November 3-6, 2019. Westin Gaslamp Hotel 11/3-6/2019 65th Annual Meeting of the Radiation Research Society, San Diego, CA, November 3-6, 2019. Poster # PS8-33. , Nov-2019
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Abstracts for Journals and Proceedings
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Nelson G, Jones T, Stanbouly S, Tolan B, Wroe A, Rosi S, Grue K, Hartman R. "Dose rate effects of protons on mouse central nervous system." 66th Annual Meeting of Radiation Research Society, Virtual Meeting, October 18-21, 2020. 66th Annual Meeting of Radiation Research Society, Virtual Meeting, October 18-21, 2020. Poster # PS9-06. , Oct-2020
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Abstracts for Journals and Proceedings
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Nelson G, Jones T, Stanbouly S, Tolan B, Wroe A, Hartman R. "Dose Rate Effects on CNS Responses to Protons: Initial Observations." 2020 NASA Human Research Program Investigators’ Workshop, Galveston, Texas, January 27-30, 2020. Abstracts. 2020 NASA Human Research Program Investigators’ Workshop, Galveston, Texas, January 27-30, 2020. , Jan-2020
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