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.

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.

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.

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.

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 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.

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.

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.

Hippocampal neurogenesis and PSA-NCAM expression following exposure to 56Fe particles mimics that seen during aging in rats.

Exposure to particles of high energy and charge can disrupt the neuronal systems as well as the motor and cognitive behaviors mediated by these systems in a similar fashion to that seen during the aging process. In the hippocampus, adult neurogenesis is affected both by aging and irradiation with ionizing particles. Likewise, the maturation of newly formed cells in this region as measured by PSA-NCAM expression is also altered by the aging process. The present study was designed to investigate the effects of 2.5 Gy of 1 GeV/n (56)Fe particles on neurogenesis using the nuclear proliferation marker 5-bromodeoxyuridine (BrdU and PSA-NCAM expression in the dentate gyrus of rats exposed to whole-body irradiation or simply placed in the chamber without being irradiated. All subjects (n=10) were sacrificed 28 days after the last BrdU injection (50 mg/kg X 3 days) and their brains were processed for immunohistochemistry. Results illustrate a decrease in the number of BrdU-positive cells as well as different distribution of these cells in the dentate gyrus of irradiated animals. Additionally, irradiated subjects show decreased levels of PSA-NCAM expression. These changes are consistent with those found in aged subjects indicating that heavy-particle irradiation is an adequate model for the study of aging.

A longitudinal study of operant responding in rats irradiated when 2 months old.

Two-month-old rats were exposed to 56Fe particles (1, 1.5, 2 Gy; 1 GeV/nucleon). They were tested on the performance of an ascending fixed-ratio operant task (bar pressing for food reward) at 7, 11 and 15 months after irradiation. Previous research had shown that for the same rats tested at 3 months after exposure, only the rats exposed to 2 Gy of 56Fe particles showed a significant disruption of performance compared to control (0 Gy) rats. When these rats were tested 7, 11 and 15 months after exposure, all irradiated groups showed significantly poorer performance than the controls. These results suggest that there is an interaction between irradiation and age such that 56Fe-particle-induced performance deficits can develop several months after exposure.

Effects of age and diet on the heavy particle-induced disruption of operant responding produced by a ground-based model for exposure to cosmic rays.

On missions to other planets, astronauts will be exposed to galactic cosmic rays which are composed of heavy particles (such as 56Fe) and protons. Exposure to these particles can affect the ability of rats to perform a variety of tasks, indicating that there is the possibility that the performance capabilities of astronauts may be affected. Previous research has shown that diets containing blueberry or strawberry extract can ameliorate the deficits produced by irradiation using a ground-based analog for exposure to cosmic rays. Rats were placed on diets containing 2% blueberry or strawberry extract for 2 months prior to exposure to 1.5 Gy of 1 GeV/n 56Fe particles. There were no effects on performance of any group of animals when tested on an ascending fixed-ratio operant task 6 months following exposure. When tested 12 months after exposure, the performance of the radiated animals given blueberry extract did not differ from the radiated animals fed the control diet. Both groups performed significantly poorer than the non-irradiated controls. There were no differences between the non-irradiated animals fed control diet and the radiated animals fed the strawberry diet and their performance was significantly better than of the radiated rats fed the blueberry or control diets. The results indicate that diets containing strawberry extract may provide a significant level of radiation protection on exploratory class missions.

Effect of diet on the disruption of operant responding at different ages following exposure to (56)Fe particles.

Rats were exposed to 2.0 Gy of (56)Fe particles to study the relationship between age and diet in the heavy particle-induced disruption of performance on an ascending fixed-ratio task. Irradiation produced a disruption of operant responding in rats tested 5 and 8 months after exposure, which was prevented by maintaining the rats on a diet containing strawberry, but not blueberry, extract. When tested 13 and 18 months after irradiation there were no differences in performance between the radiated rats maintained on control, strawberry or blueberry diets. These observations suggest that the beneficial effects of antioxidant diets may be dependent upon the age of testing.

Diet as a factor in behavioral radiation protection following exposure to heavy particles.

Major risks associated with radiation exposures on deep space missions include carcinogenesis due to heavy-particle exposure of cancer-prone tissues and performance decrements due to neurological damage produced by heavy particles. Because exposure to heavy particles can cause oxidative stress, it is possible that antioxidants can be used to mitigate these risks (and possibly some health risks of microgravity). To assess the capacity of antioxidant diets to mitigate the effects of exposure to heavy particles, rats were maintained on antioxidant diets containing 2% blueberry or strawberry extract or a control diet for 8 weeks prior to exposure to 1.5 or 2.0 Gy of accelerated iron particles at Brookhaven National Laboratory. Following irradiation rats were tested on a series of behavioral tasks: amphetamine-induced taste aversion learning, operant responding and spatial learning and memory. The results indicated that the performance of the irradiated rats maintained on the antioxidant diets was, in general, significantly better than that of the control animals, although the effectiveness of the diets ameliorating the radiation-induced deterioration in performance varied as a function of both the specific diet and the specific endpoint. In addition, animals fed antioxidant diets prior to exposure showed reduced heavy particle-induced tumorigenesis one year after exposure compared to the animals fed the control diet. These results suggest that antioxidant diets have the potential to serve as part of a system designed to provide protection to astronauts against the effects of heavy particles on exploratory missions outside the magnetic field of the earth.

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.

Effects of heavy particle irradiation and diet on amphetamine- and lithium chloride-induced taste avoidance learning in rats.

Rats were maintained on diets containing either 2% blueberry or strawberry extract or a control diet for 8 weeks prior to being exposed to 1.5 Gy of 56Fe particles in the Alternating Gradient Synchrotron at Brookhaven National Laboratory. Three days following irradiation, the rats were tested for the effects of irradiation on the acquisition of an amphetamine- or lithium chloride-induced (LiCl) conditioned taste avoidance (CTA). The rats maintained on the control diet failed to show the acquisition of a CTA following injection of amphetamine. In contrast, the rats maintained on antioxidant diets (strawberry or blueberry extract) continued to show the development of an amphetamine-induced CTA following exposure to 56Fe particles. Neither irradiation nor diet had an effect on the acquisition of a LiCl-induced CTA. The results are interpreted as indicating that oxidative stress following exposure to 56Fe particles may be responsible for the disruption of the dopamine-mediated amphetamine-induced CTA in rats fed control diets; and that a reduction in oxidative stress produced by the antioxidant diets functions to reinstate the dopamine-mediated CTA. The failure of either irradiation or diet to influence LiCl-induced responding suggests that oxidative stress may not be involved in CTA learning following injection of LiCl.

Effects of exposure to 56Fe particles or protons on fixed-ratio operant responding in rats.

On long-duration trips outside of the magnetosphere, astronauts will be exposed to protons and to heavy particles which can affect their performance of required tasks. It is essential to determine the range of behaviors that might be affected by exposure to these types of radiation in order to understand the nature of behavioral deficits and to develop effective countermeasures. The present experiment examined the ability of rats to make an operant response following exposure to protons (250 MeV, 4 Gy) or 56Fe particles (1 GeV/n, 1 or 2 Gy). Following irradiation, rats were trained to press a lever in order to obtain food reinforcement. They were then placed on an ascending fixed-ratio schedule from FR-1 (each lever press rewarded with a food pellet) through FR-35 (35 lever presses required for 1 food pellet). Rats exposed to 4 Gy of protons or 1 Gy of 56Fe particles responded similarly to controls, increasing their rate of responding as the ratio increased. However, rats exposed to 2 Gy of 56Fe particles failed to increase their rate of responding at ratios greater than FR-20, indicating that rats exposed to 2 Gy of 56Fe particles cannot respond appropriately to increasing work requirements.