Kirsty L. Carrihill-Knoll

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 (16O) (1000 MeV/n). Compared to nonirradiated controls, exposure to 16O 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 16O-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 16O 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 16O-particle radiation on oxidative stress, inflammation and loss of autophagy in the brain of young rats.

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

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.

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.

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 56Fe 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.

Comparison of the Effectiveness of Exposure to Low-LET Helium Particles (4He) and Gamma Rays (137Cs) 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 (4He) particles (1,000 MeV/n; 0.1–10 cGy; head only) or cesium 137Cs gamma rays (50–400 cGy; whole body), after which their cognitive performance was evaluated. The results indicated that exposure to doses of 4He 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 137Cs 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 56Fe and 16O 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 (16O and 56Fe) 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 56Fe and 16O are widespread, only changes in specific regions of the brain may be related to changes in cognitive function.

Neuronal stress following exposure to 56Fe particles and the effects of antioxidant-rich diets

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 and disrupts the functioning of neuronal communication in critical regions of the brain [ 1]. These changes in neuronal function are similar to those seen in ageing [ 2, 3]. Although there is some recovery of function after exposure to 56Fe particles, particularly in changes observed 36 h following irradiation, long-term changes (75 days) have been observed, suggesting subcellular damage. Consequently, oxidative stress and inflammation induced by radiation could affect downstream events, such as changes in behavior and gene expression. Therefore, berry fruits high in antioxidant and anti-inflammatory activity, such as blueberries and strawberries, may prevent the occurrence of neurochemical and behavioral changes that occur if fed prior to radiation [ 4]. Rats were exposed to 56Fe (1000 MeV/n; 1.5 Gy) particles at the NASA Space Radiation Laboratory at Brookhaven National Laboratory; other rats served as non-irradiated controls. The animals were fed either a control or a 2% blueberry or strawberry diet 8 weeks prior to radiation. Rats were then either euthanized at 36 h (short term) or 30 days following irradiation (long term). Before and after the irradiation, the animals were housed at USDA Human Nutrition Research Center on Aging at Tufts University, Boston. The results of the experiments indicate that: (1) 56Fe exposure caused significant differential, neurochemical changes in critical regions of the brain, such as hippocampus, striatum, frontal cortex and cerebellum, particularly long term. (2) Neurochemical changes resulted in the disruption of autophagy, increased inflammation and increased oxidative stress protein markers. (3) Antioxidant-rich berry diets significantly reduced the accumulation of toxic cellular debris in critical regions of the brain, primarily at the 30 days post-irradiation time-point. (4) Susceptibility to inflammation, autophagy dysregulation, and oxidative stress were proportional to the levels of antioxidant enzymes in the respective brain regions. (5) Exposure to 56Fe radiation may cause the accumulation of disease-related proteins such as PHF-Tau, which has been implicated in the pathogenesis of Alzheimers disease. Irradiation with 56Fe, which causes substantial build-up of toxic cellular debris in critical regions of the brain, may overwhelm the innate antioxidant enzyme defense system [ 5]. Therefore, berry diets high in antioxidants may be used to counter these damaging effects by reducing oxidative stress and inflammation, and activating neuronal housekeeping, in addition to boosting endogenous antioxidant enzymes. This paper was presented at the NASA Session at Heavy Ion in Therapy and Space Radiation Symposium 2013.

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.

Cognitive effects of partial and whole-body exposures to 16O particles

Introduction: When rats and mice are exposed to HZE particles at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory to simulate the effect of exposure to space radiation on cognitive performance, there may be differences in the amount of tissue that is irradiated: some experimenters irradiate only the head, whereas others irradiate the entire organism. Whether or not these different patterns of exposure have differential effects on cognitive performance remain to be established. Similarly, it remains to be established whether or not exposures restricted to the body can have an independent effect on cognitive performance. Radiation: Rats were exposed to 16O (1000 MeV/n; 1, 5, 10, 25 cGy) particles at the NSRL. There were three exposure conditions: head-only, body-only and whole body. Tungsten bricks were used to shield either the head or the body, as required. The bricks were removed for whole-body exposures. The non-irradiated control rats (0 cGy) were taken to the NSRL, but not exposed. Nominal dose rates were between 1 and 10 cGy/min, depending upon the total dose. Behavior: The animals were shipped to UMBC for behavioral testing. Cognitive performance was measured using the elevated-plus-maze (baseline anxiety); novel object recognition (general learning and memory); novel spatial location (spatial learning and memory); and operant responding on an ascending fixed-ratio schedule (motivation and responsiveness to environmental stimuli). The basis for the selection of the behavioral tests was that the behaviors are dopamine-mediated or show deterioration as a function of age. Results: The results of the experiments indicate that: (i) there may be differences in cognitive performance following exposure to either head-only or whole-body exposure to NASA-relevant doses of 16O particles depending on the specific task; and (ii) for some tasks, body-only exposures can disrupt neurocognitive performance. The results also suggest that exposures involving the body may influence the responsiveness of the organism to the disruptive effects of exposure to HZE particles on neurocognitive performance. This effect is seen in two ways: first, as a direct effect of body-only exposure on cognitive performance, as shown by decreased time spent in a novel location compared with non-irradiated controls, suggesting impaired spatial location memory; and second, as an interaction between head and body exposures seen with whole-body exposures. Discussion: These results indicate that body-only exposures have the potential to influence cognitive performance which is mediated by the brain. Although the mechanism remains to be fully established, it is possible that exposure of the body to HZE particles causes the release of cytokines which can affect neuronal function, either directly or through the mediation of the vagus nerve. As such, the results of studies using head-only or whole-body exposures may not be comparable. Also, astronauts may be subject to increased risk of deficits in cognitive performance during exploratory class missions.