Galactic Cosmic Ray Particle Exposure Does Not Increase Protein Levels of Inflammation or Oxidative Stress Markers in Rat Microglial Cells In Vitro.

Astronauts on exploratory missions will be exposed to galactic cosmic rays (GCR), which can induce neuroinflammation and oxidative stress (OS) and may increase the risk of neurodegenerative disease. As key regulators of inflammation and OS in the CNS, microglial cells may be involved in GCR-induced deficits, and therefore could be a target for neuroprotection. This study assessed the effects of exposure to helium (4He) and iron (56Fe) particles on inflammation and OS in microglia in vitro, to establish a model for testing countermeasure efficacy. Rat microglia were exposed to a single dose of 20 cGy (300 MeV/n) 4He or 2 Gy 56Fe (600 MeV/n), while the control cells were not exposed (0 cGy). Immediately following irradiation, fresh media was applied to the cells, and biomarkers of inflammation (cyclooxygenase-2 [COX-2], nitric oxide synthase [iNOS], phosphorylated IκB-α [pIκB-α], tumor necrosis factor-α [TNFα], and nitrite [NO2-]) and OS (NADPH oxidase [NOX2]) were assessed 24 h later using standard immunochemical techniques. Results showed that radiation did not increase levels of NO2- or protein levels of COX-2, iNOS, pIκB-α, TNFα, or NOX2 compared to non-irradiated control conditions in microglial cells (p > 0.05). Therefore, microglia in isolation may not be the primary cause of neuroinflammation and OS following exposures to helium or iron GCR particles.

Effects of HZE-Particle Exposure Location and Energy on Brain Inflammation and Oxidative Stress in Rats.

Astronauts on exploratory missions will be exposed to particle radiation of high energy and charge (HZE particles), which have been shown to produce neurochemical and performance deficits in animal models. Exposure to HZE particles can produce both targeted effects, resulting from direct ionization of atoms along the particle track, and non-targeted effects (NTEs) in cells that are distant from the track, extending the range of potential damage beyond the site of irradiation. While recent work suggests that NTEs are primarily responsible for changes in cognitive function after HZE exposures, the relative contributions of targeted and non-targeted effects to neurochemical changes after HZE exposures are unclear. The present experiment was designed to further explore the role of targeted and non-targeted effects on HZE-induced neurochemical changes (inflammation and oxidative stress) by evaluating the effects of exposure location and particle energy/linear energy transfer (LET). Forty-six male Sprague-Dawley rats received head-only or body-only exposures to 56Fe particles [600 MeV/n (75 cGy) or 1,000 MeV/n (100 cGy)] or 48Ti particles [500 MeV/n (50 cGy) or 1,100 MeV/n (75 cGy)] or no irradiation (0 cGy). Twenty-four h after irradiation, rats were euthanized, and the brain was dissected for analysis of HZE-particle-induced neurochemical changes in the hippocampus and frontal cortex. Results showed that exposure to 56Fe and 48Ti ions produced changes in measurements of brain inflammation [glial fibrillary astrocyte protein (GFAP)], oxidative stress [NADPH-oxidoreductase-2 (NOX2)] and antioxidant enzymes [superoxide dismutase (SOD), glutathione S-transferase (GST), nuclear factor erythroid 2-related factor 2 (Nrf2)]. However, radiation effects varied depending upon the specific measurement, brain region, and exposure location. Although overall exposures of the head produced more detrimental changes in neuroinflammation and oxidative stress than exposures of the body, body-only exposures also produced changes relative to no irradiation, and the effect of particle energy/LET on neurochemical changes was minimal. Results indicate that both targeted and non-targeted effects are important contributors to neurochemical changes after head-only exposure. However, because there were no consistent neurochemical changes as a function of changes in track structure after head-only exposures, the role of direct effects on neuronal function is uncertain. Therefore, these findings, although in an animal model, suggest that NTEs should be considered in the estimation of risk to the central nervous system (CNS) and development of countermeasures.

Effects of preexposure to a subthreshold dose of helium particles on the changes in performance produced by exposure to helium particles.

On exploratory class missions, such as a mission to Mars, astronauts will be exposed to doses of particles of high energy and charge and protons up to 30 - 40 cGy. These exposures will most likely occur at random intervals across the estimated 3-yr duration of the mission. As such, the possibility of an interaction between particles must be taken into account: a prior subthreshold exposure to one particle may prevent or minimize the effect of a subsequent exposure (adaptation), or there may be an additive effect such that the prior exposure may sensitize the individual to a subsequent exposure of the same or different radiations. Two identical replications were run in which rats were exposed to a below threshold dose of 4He particles and 2, 24 or 72 h later given either a second below threshold or an above threshold dose of 4He particles and tested for performance on an operant task. The results indicate that preexposure to a subthreshold dose of 4He particles can either sensitize or attenuate the effects of the subsequent dose, depending upon the interval between exposures and the doses. These results suggest that exposure to multiple doses of heavy particles may have implications for astronaut health on exploratory class missions.

Modeling space radiation induced cognitive dysfunction using targeted and non-targeted effects.

Radiation-induced cognitive dysfunction is increasingly recognized as an important risk for human exploration of distant planets. Mechanistically-motivated mathematical modeling helps to interpret and quantify this phenomenon. Here we considered two general mechanisms of ionizing radiation-induced damage: targeted effects (TE), caused by traversal of cells by ionizing tracks, and non-targeted effects (NTE), caused by responses of other cells to signals released by traversed cells. We compared the performances of 18 dose response model variants based on these concepts, fitted by robust nonlinear regression to a large published data set on novel object recognition testing in rats exposed to multiple space-relevant radiation types (H, C, O, Si, Ti and Fe ions), covering wide ranges of linear energy transfer (LET) (0.22-181 keV/µm) and dose (0.001-2 Gy). The best-fitting model (based on Akaike information criterion) was an NTE + TE variant where NTE saturate at low doses (~ 0.01 Gy) and occur at all tested LETs, whereas TE depend on dose linearly with a slope that increases with LET. The importance of NTE was also found by additional analyses of the data using quantile regression and random forests. These results suggest that NTE-based radiation effects on brain function are potentially important for astronaut health and for space mission risk assessments.

Effects of partial- or whole-body exposures to 56Fe particles on brain function and cognitive performance in rats.

On exploratory class missions, such as a mission to Mars, astronauts will be exposed to particles of high energy and charge (HZE particles). Exposure to HZE particles produces changes in neuronal function and can disrupt cognitive performance. Cells throughout the entire body, not just the brain, will be impacted by these particles. To determine the possible effects that irradiation of the body might have on neuronal function and cognitive performance, rats were given head-only, body-only or whole-body exposures to 56Fe particles. Cognitive performance (novel object recognition, operant responding) was tested in one set of animals; changes in brain function (oxidative stress, neuroinflammation) was tested in a second set of rats. The results indicated that there were no consistent differences in either behavioral or neurochemical endpoints as a function of the location of the irradiation. These results suggest that radiation to the body can impact the brain, therefore it may be necessary to re-evaluate the estimates of the risk of HZE particle-induced changes in neuronal function and cognitive performance.

Effects of exposure to 12C and 4He particles on cognitive performance of intact and ovariectomized female rats.

Exposure to the types of radiation encountered outside the magnetic field of the earth can disrupt cognitive performance. Exploratory class missions to other planets will include both male and female astronauts. Because estrogen can function as a neuroprotectant, it is possible that female astronauts may be less affected by exposure to space radiation than male astronauts. To evaluate the effectiveness of estrogen to protect against the disruption of cognitive performance by exposure to space radiation intact and ovariectomized female rats with estradiol or vehicle implants were tested on novel object performance and operant responding on an ascending fixed-ratio reinforcement schedule following exposure to 12C (290 MeV/n) or 4He (300 MeV/n) particles. The results indicated that exposure to carbon or helium particles did not disrupt cognitive performance in the intact rats. Estradiol implants in the ovariectomized subjects exacerbated the disruptive effects of space radiation on operant performance. Although estrogen does not appear to function as a neuroprotectant following exposure to space radiation, the present data suggest that intact females may be less responsive to the deleterious effects of exposure to space radiation on cognitive performance, possibly due to the effects of estrogen on cognitive performance.

Effects of head-only or whole-body exposure to very low doses of 4He (1000 MeV/n) particles on neuronal function and cognitive performance.

On exploratory class missions, astronauts will be exposed to a range of heavy particles which vary in linear energy transfer (LET). Previous research has shown a direct relationship between particle LET and cognitive performance such that, as particle LET decreases the dose needed to affect cognitive performance also decreases. Because a significant portion of the total dose experienced by astronauts may be expected to come from exposure to low LET 4He particles, it would be important to establish the threshold dose of 4He particles that can produce changes in cognitive performance. The results indicated that changes in neuronal function and cognitive performance could be observed following both head-only and whole-body exposures to 4He particles at doses as low as 0.01-0.025 cGy. These results, therefore, suggest the possibility that astronauts on exploratory class missions may be at a greater risk for HZE-induced deficits than previously anticipated.

Age as a factor in the responsiveness of the organism to the disruption of cognitive performance by exposure to HZE particles differing in linear energy transfer.

Exposure to particles of high energy and charge (HZE particles) can produce decrements in cognitive performance. A series of experiments exposing rats to different HZE particles was run to evaluate whether the performance decrement was dependent on the age of the subject at the time of irradiation. Fischer 344 rats that were 2-, 11- and 15/16-months of age were exposed to 16O, 48Ti, or 4He particles at the NASA Space Radiation Laboratory at Brookhaven National Laboratory. As previously observed following exposure to 56Fe particles, exposure to the higher LET 48Ti particles produced a disruption of cognitive performance at a lower dose in the older subjects compared to the dose needed to disrupt performance in the younger subjects. There were no age related changes in the dose needed to produce a disruption of cognitive performance following exposure to lower LET 16O or 4He particles. The threshold for the rats exposed to either 16O or 4He particles was similar at all ages. Because the 11- and 15-month old rats are more representative of the age of astronauts (45-55 years old) the present results indicate that particle LET may be a critical factor in estimating the risk of developing a cognitive deficit following exposure to space radiation on exploratory class missions.

Neurochemical differences in learning and memory paradigms among rats supplemented with anthocyanin-rich blueberry diets and exposed to acute doses of 56Fe particles.

The protective effects of anthocyanin-rich blueberries (BB) on brain health are well documented and are particularly important under conditions of high oxidative stress, which can lead to "accelerated aging." One such scenario is exposure to space radiation, consisting of high-energy and -charge particles (HZE), which are known to cause cognitive dysfunction and deleterious neurochemical alterations. We recently tested the behavioral and neurochemical effects of acute exposure to HZE particles such as 56Fe, within 24-48h after exposure, and found that radiation primarily affects memory and not learning. Importantly, we observed that specific brain regions failed to upregulate antioxidant and anti-inflammatory mechanisms in response to this insult. To further examine these endogenous response mechanisms, we have supplemented young rats with diets rich in BB, which are known to contain high amounts of antioxidant-phytochemicals, prior to irradiation. Exposure to 56Fe caused significant neurochemical changes in hippocampus and frontal cortex, the two critical regions of the brain involved in cognitive function. BB supplementation significantly attenuated protein carbonylation, which was significantly increased by exposure to 56Fe in the hippocampus and frontal cortex. Moreover, BB supplementation significantly reduced radiation-induced elevations in NADPH-oxidoreductase-2 (NOX2) and cyclooxygenase-2 (COX-2), and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) in the hippocampus and frontal cortex. Overall results indicate that 56Fe particles may induce their toxic effects on hippocampus and frontal cortex by reactive oxygen species (ROS) overload, which can cause alterations in the neuronal environment, eventually leading to hippocampal neuronal death and subsequent impairment of cognitive function. Blueberry supplementation provides an effective preventative measure to reduce the ROS load on the CNS in an event of acute HZE exposure.

Comparison of the Effectiveness of Exposure to Low-LET Helium Particles ((4)He) and Gamma Rays ((137)Cs) on the Disruption of Cognitive Performance.

In this study, the effects of radiation exposure on cognitive performance were evaluated. Rats were exposed to either helium ((4)He) particles (1,000 MeV/n; 0.1-10 cGy; head only) or cesium (137)Cs gamma rays (50-400 cGy; whole body), after which their cognitive performance was evaluated. The results indicated that exposure to doses of (4)He particles as low as 0.1 cGy disrupted performance in a variety of cognitive tasks, including plus-maze performance (baseline anxiety), novel location recognition (spatial performance) and operant responding on an ascending fixed-ratio reinforcement schedule (motivation and responsiveness to changes in environmental contingencies) but not on novel object recognition performance (learning and memory). In contrast, after exposure to (137)Cs gamma rays only plus-maze performance was affected. There were no significant effects on any other task. Because exposure to both types of radiation produce oxidative stress, these results indicate that radiation-produced oxidative stress may be a necessary condition for the radiation-induced disruption of cognitive performance, but it is not a sufficient condition.

Acute Effects of Exposure to (56)Fe and (16)O Particles on Learning and Memory.

Although it has been shown that exposure to HZE particles disrupts cognitive performance when tested 2-4 weeks after irradiation, it has not been determined whether exposure to HZE particles acutely affects cognitive performance, i.e., within 4-48 h after exposure. The current experiments were designed to determine the acute effects of exposure to HZE particles ((16)O and (56)Fe) on cognitive performance and whether exposure to HZE particles affected learning or memory, as well as to understand the relationship between acute changes in the levels of NOX2 (a measure of oxidative stress) and COX2 (a measure of neuroinflammation) in specific brain regions and cognitive performance. The results of these studies indicate that the acute effects of radiation exposure on cognitive performance are on memory, not learning. Further, the acute effects of exposure to HZE particles on oxidative stress and neuroinflammation and their relationship to cognitive performance indicate that, although the effects of exposure to both (56)Fe and (16)O are widespread, only changes in specific regions of the brain may be related to changes in cognitive function.

Lack of reliability in the disruption of cognitive performance following exposure to protons.

A series of three replications were run to determine the reliability with which exposure to protons produces a disruption of cognitive performance, using a novel object recognition task and operant responding on an ascending fixed-ratio task. For the first two replications, rats were exposed to head-only exposures to 1000 MeV/n protons at the NASA Space Radiation Laboratory. For the third replication, subjects were given head-only or whole-body exposures to both 1000 and 150 MeV/n protons. The results were characterized by a lack of consistency in the effects of exposure to protons on the performance of these cognitive tasks, both within and between replications. The factors that might influence the lack of consistency and the implications for exploratory class missions are discussed.

Protective effects of blueberry- and strawberry diets on neuronal stress following exposure to (56)Fe particles.

Particles of high energy and charge (HZE particles), which are abundant outside the magnetic field of the Earth, have been shown to disrupt the functioning of neuronal communication in critical regions of the brain. Previous studies with HZE particles, have shown that irradiation produces enhanced indices of oxidative stress and inflammation as well as altered neuronal function that are similar to those seen in aging. Feeding animals antioxidant-rich berry diets, specifically blueberries and strawberries, countered the deleterious effects of irradiation by reducing oxidative stress and inflammation, thereby improving neuronal signaling. In the current study, we examined the effects of exposure to (56)Fe particles in critical regions of brain involved in cognitive function, both 36h and 30 days post irradiation. We also studied the effects of antioxidant-rich berry diets, specifically a 2% blueberry or strawberry diet, fed for 8 weeks prior to radiation as well as 30 days post irradiation. (56)Fe exposure caused significant differential, neurochemical changes in critical regions of the brain, such as hippocampus, striatum, frontal cortex, and cerebellum, through increased inflammation, and increased oxidative stress protein markers. (56)Fe exposure altered the autophagy markers, and antioxidant-rich berry diets significantly reduced the accumulation of p62 in hippocampus, a scaffold protein that co-localizes with ubiquitinated protein at the 30 days post irradiation time-point. Exposure to (56)Fe particles increased the accumulation of disease-related proteins such as PHF-tau in the hippocampus of animals fed the control diet, but not in the irradiated animals fed the blueberry diet. These results indicate the potential protective effects of antioxidant-rich berry diets on neuronal functioning following exposure to HZE particles.

Comparison of the effects of partial- or whole-body exposures to ¹6O particles on cognitive performance in rats.

Studies using a ground-based system (NASA Space Radiation Laboratory) to examine the effects of exposure to high-energy charged particles or HZE particles on cognitive performance have interchangeably used whole-body exposures or exposures restricted to the head of the subject. For this study, we hypothesized that different types of exposure such as whole body vs. head only vs. body only might modulate the impact of irradiation on cognitive performance in different ways with the resulting cognitive performance outcomes being either independent of exposure type or strongly dependent on exposure type with each producing performance outcomes. To test these possibilities, three groups of rats were exposed to ¹6O particles (1,000 MeV/n): (1) head only; (2) body only; (3) whole body. Cognitive performance was measured using the elevated plus-maze, novel object recognition, spatial location memory and operant responding on an ascending fixed-ratio schedule. The results indicated that the performance of the rats on the spatial location memory task was markedly different when they received head-only irradiation compared to whole-body exposure. For the operant responding task, irradiation of the whole body resulted in a more severe performance decrement than exposures restricted to the head. The results are discussed in terms of nontargeted effects of HZE particles and the findings suggest that studies that utilize different patterns of exposure may not be directly comparable and that astronauts may be at a greater risk for HZE particle-induced cognitive deficits than previously thought.

Changes in gene expression in the rat hippocampus following exposure to 56Fe particles and protection by berry diets.

Exposing young rats to particles of high energy and charge, such as (56)Fe, enhances indices of oxidative stress and inflammation and disrupts behavior, including spatial learning and memory. In the present study, we examined whether gene expression in the hippocampus, an area of the brain important in memory, is affected by exposure to 1.5 Gy or 2.5 Gy of 1 GeV/n high-energy (56)Fe particles 36 hours after irradiation. We also determined if 8 weeks of pre-feeding with 2% blueberry or 2% strawberry antioxidant diets could ameliorate irradiation-induced changes in gene expression. Alterations in gene expression profile were analyzed by pathway-focused microarrays for inflammatory cytokines and genes involved in nuclear factor-kappa B (NF-κB) signal transduction pathways. We found that genes that are directly or indirectly involved in the regulation of growth and differentiation of neurons were changed following irradiation. Genes that regulate apoptosis were up-regulated whereas genes that modulate cellular proliferation were down-regulated. The brains of animals supplemented with berry diets demonstrated an up-regulation of some protective stress signal genes. Therefore, these data suggest that (56)Fe particle irradiation causes changes in gene expression in rats that are ameliorated by berry fruit diets.

Interaction between age of irradiation and age of testing in the disruption of operant performance using a ground-based model for exposure to cosmic rays.

Previous research has shown a progressive deterioration in cognitive performance in rats exposed to (56)Fe particles as a function of age. The present experiment was designed to evaluate the effects of age of irradiation independently of the age of testing. Male Fischer-344 rats, 2, 7, 12, and 16 months of age, were exposed to 25-200 cGy of (56)Fe particles (1,000 MeV/n). Following irradiation, the rats were trained to make an operant response on an ascending fixed-ratio reinforcement schedule. When performance was evaluated as a function of both age of irradiation and testing, the results showed a significant effect of age on the dose needed to produce a performance decrement, such that older rats exposed to lower doses of (56)Fe particles showed a performance decrement compared to younger rats. When performance was evaluated as a function of age of irradiation with the age of testing held constant, the results indicated that age of irradiation was a significant factor influencing operant responding, such that older rats tested at similar ages and exposed to similar doses of (56)Fe particles showed similar performance decrements. The results are interpreted as indicating that the performance decrement is not a function of age per se, but instead is dependent upon an interaction between the age of irradiation, the age of testing, and exposure to HZE particles. The nature of these effects and how age of irradiation affects cognitive performance after an interval of 15 to 16 months remains to be established.

Exposure to 16O-particle radiation causes aging-like decrements in rats through increased oxidative stress, inflammation and loss of autophagy.

Exposing young rats to particles of high energy and charge (HZE particles), a ground-based model for exposure to cosmic rays, enhances indices of oxidative stress and inflammation, disrupts the functioning of neuronal communication, and alters cognitive behaviors. Even though exposure to HZE particles occurs at low fluence rates, the cumulative effects of long-term exposure result in molecular changes similar to those seen in aged animals. In the present study, we assessed markers of autophagy, a dynamic process for intracellular degradation and recycling of toxic proteins and organelles, as well as stress and inflammatory responses, in the brains of Sprague-Dawley rats irradiated at 2 months of age with 5 and 50 cGy and 1 Gy of ionizing oxygen particles ((16)O) (1000 MeV/n). Compared to nonirradiated controls, exposure to (16)O particles significantly inhibited autophagy function in the hippocampus as measured by accumulation of ubiquitin inclusion bodies such as P62/SQSTM1, autophagosome marker microtubule-associated protein 1 beta light chain 3 (MAP1B-LC3), beclin1 and proteins such as mammalian target of rapamycin (mTOR). The molecular changes measured at short (36 h) and long (75 days) intervals after (16)O-particle exposure indicate that the loss of autophagy function occurred shortly after exposure but was recovered via inhibition of mTOR. However, HZE-particle radiation caused significant sustained loss of protein kinase C alpha (PKC-α), a key G protein modulator involved in neuronal survival and functions of neuronal trophic factors. Exposure to (16)O particles also caused substantial increases in the levels of nuclear factor kappa B (NF-κB) and glial fibrillary acidic protein (GFAP), indicating glial cell activation 75 days after exposure. This is the first report to show the molecular effects of (16)O-particle radiation on oxidative stress, inflammation and loss of autophagy in the brain of young rats.

In vivo space radiation-induced non-targeted responses: late effects on molecular signaling in mitochondria.

The lack of clear knowledge about space radiation-induced biological effects has been singled out as the most important factor limiting the prediction of radiation risk associated with human space exploration. The expression of space radiation-induced non-targeted effects is thought to impact our understanding of the health risks associated with exposure to low fluences of particulate radiation encountered by astronauts during prolonged space travel. Following a brief review of radiation-induced bystander effects and the growing literature for the involvement of oxidative metabolism in their expression, we show novel data on the induction of in vivo non-targeted effects following exposure to 1100 MeV/nucleon titanium ions. Analyses of proteins by two-dimensional gel electrophoresis in non-targeted liver of cranially-irradiated Sprague Dawley rats revealed that the levels of key proteins involved in mitochondrial fatty acid metabolism are decreased. In contrast, those of proteins involved in various cellular defense mechanisms, including antioxidation, were increased. These data contribute to our understanding of the mechanisms underlying the biological responses to space radiation, and support the involvement of mitochondrial processes in the expression of radiation induced non-targeted effects. Significantly, they reveal the cross-talk between propagated stressful effects and induced adaptive responses. Together, with the accumulating data in the field, our results may help reduce the uncertainty in the assessment of the health risks to astronauts. They further demonstrate that 'network analyses' is an effective tool towards characterizing the signaling pathways that mediate the long-term biological effects of space radiation.

Beneficial effects of fruit extracts on neuronal function and behavior in a rodent model of accelerated aging.

Exposing young rats to particles of high-energy and charge (HZE particles) enhances indices of oxidative stress and inflammation and disrupts the functioning of the dopaminergic system and behaviors mediated by this system in a manner similar to that seen in aged animals. Previous research has shown that diets supplemented with 2% blueberry or strawberry extracts have the ability to retard and even reverse age-related deficits in behavior and signal transduction in rats, perhaps due to their antioxidant and anti-inflammatory properties. This study evaluated the efficacy of these diets on irradiation-induced deficits in these parameters by maintaining rats on these diets or a control diet for 8 weeks prior to being exposed to whole-body irradiation with 1.5 Gy of 1 GeV/n high-energy (56)Fe particles. Irradiation impaired performance in the Morris water maze and measures of dopamine release 1 month following radiation; these deficits were protected by the antioxidant diets. The strawberry diet offered better protection against spatial deficits in the maze because strawberry-fed animals were better able to retain place information (a hippocampally mediated behavior) compared to controls. The blueberry diet, on the other hand, seemed to improve reversal learning, a behavior more dependent on intact striatal function. These data suggest that (56)Fe particle irradiation causes deficits in behavior and signaling in rats which were ameliorated by an antioxidant diet and that the polyphenols in these fruits might be acting in different brain regions.