This hypothesis was explored in the following four specific aims:

Aim 1. We will establish dose and time response patterns and explore interactions among inflammatory changes in brain, lung, and the systemic circulation following whole body 1000 MeV/n proton irradiation of C57BL/6 mice using the following mission-relevant doses: 200, 100, 50, 25, and 10 cGy. Similar measures will be obtained for mice exposed to mixed proton and HZE particles as well as a simulated solar particle event.

Aim 2. We will carry out analyses of hippocampal neurogenesis and hippocampal dependent and independent learning up to one year following radiation exposure and correlate changes with measures of inflammation.

Aim 3. We will determine whether space radiation exposure impacts neurodegenerative disease pathogenesis by exposing a transgenic mouse model of Alzheimer’s disease to a subset of these treatments. Specific outcomes include extent of tissue inflammation, AD-related pathology, and behavioral changes. Note that the model chosen is distinct from that being tested in the current CNS NSCOR and therefore offers a complementary means to explore this critical issue.

Aim 4. We will determine whether space radiation induced inflammation enhances the lung’s response to subsequent pulmonary challenge with lipopolysaccharide, a model of pulmonary infection.

These aims remained essentially unchanged in the course of the grant period. During this grant we conducted six separate runs at NSRL:

• Dose and time effects of protons on inflammatory indices in brain and lung (Aim 1, Experiment 1.1) and on neurogenesis and hippocampal dependent learning and memory (Aim 2, Experiment 2.1): May 4-7, 2009; NSRL Run 09A; 576 total mice used.

• Sex Differences (Experiments 1.2 and 2.2) and Mixed Particle Exposure (Experiments 1.3 and 2.3): November 17-20, 2009; NSRL Run 09C; 750 mice used.

• Proton-LPS interactions (Aim 4, Experiment 4): May 5-7, 2010; NSRL Run 10A; 150 mice used.

• HZE Effects in Alzheimer’s transgenic mice (Aim 3, Experiment 3): May 12-13, 2011; NSRL Run 11A; 75 mice used.

• Solar Flare Late Effects (Experiments 1.4 and 2.4): May 16-17, 2011; NSRL Run 11A; 150 mice used.

• Solar Flare Early Effects (added on to Experiments 1.4 and 2.4): June 25, 2012; NSRL Run 12B; 50 mice used.

We completed all experiments originally proposed in the grant and have largely completed data analyses for all aims. Although we had originally planned to carry out mRNA quantification for markers of inflammation (e.g. cytokines) in brain and lung and ELISA based measures of cytokines in blood at multiple time points, the relative lack of effects seen with inflammatory markers or behavioral outcomes at later time points reduces the rationale for carrying out these measures. However, we may elect to conduct such measures for a subset of samples in order to determine if the doses of protons used in this study show an acute systemic effect (e.g. within first 24 h).

Based on the studies conducted in this grant, the following conclusions can be made:

• Acute 1000 MeV/n proton exposure and simulated solar flare exposures at doses up to 200 cGy showed no demonstrable effects on hippocampal-dependent memory as assessed by contextual fear conditioning at times ranging from 3 to 12 months post irradiation in both male and female C57BL/6 mice irradiated at 10-12 weeks of age

• These same exposures reduced acute (within first 12 h) proliferation of adult hippocampal neural precursor cells with dose effects as low at 50 cGy for male mice and 10 cGy for female mice, but had modest or no effects on long term hippocampal neurogenesis assessed by doublecortin staining

• Neuroinflammatory changes assessed by markers of glial or endothelial activation were essentially absent in brains from mice irradiated under the conditions described above

• Addition of 10 cGy HZE irradiation (1000 MeV/n iron particles) further reduced acute neural precursor proliferation, but otherwise showed no effect in other measured indices

• There was no evidence of radiation induced late effects in lung tissue or exacerbation of inhaled LPS exposure

• APPswe/PS-1dE9 transgenic mice showed behavioral deficits and increased amyloid plaque burden following exposure to 1000 MeV/n iron particles; the later effect was observed in male mice at 9.5 months of age, 6 months post 100 cGy irradiation

We believe that these studies help to address possible thresholds underlying acute and long-term risks from space radiation (CNS Gaps 1 and 2), at least as related to adult hippocampal neurogenesis, and demonstrate that space radiation can enhance susceptibility to pathology associated with neurodegenerative disease (CNS Gap 3).

One caveat of our work was the choice of contextual fear conditioning for our behavioral assay. Our adoption of this approach was based on our previous experience exploring cognitive changes following neuroinflammation localized to the hippocampus and discussions with other NASA investigators, who had also adopted this approach for their work. More recent work suggests that other tasks may be more sensitive to radiation, including novel object recognition, which we did incorporate into some of our later studies. Selection of the proper tests for behavioral effects remains a major challenge, particularly with mice.

We are following up on this work with a currently funded NASA grant (NNX13AC33G) exploring the effects of HZE and proton exposure on Alzheimer’s pathology in a different mouse model that demonstrates both amyloid plaques and neurofibrillary tangles. A second part of that grant will examine effects of space radiation on behavior and pathology in a genetic mouse model of Parkinson’s disease. Importantly, these models show more gradual onset of disease pathology than the APP/PS1 model reported here. Therefore mice will be irradiated at 6 months of age, in accordance with guidelines to better model exposure in adulthood.

In conclusion, we have provided clear evidence of acute effects on adult hippocampal neural progenitor cells exposed to protons and enhanced Alzheimer-like pathology in a specific mouse model following HZE particle irradiation. Further studies, some of which are already in progress, will help to establish whether such changes pose a risk for space explorers.

A second series of exposures, involving nearly 750 mice was conducted as part of NSRL Run 09C. These studies essentially comprised experiments 1.2 and 2.2 (Sex Differences) as well as 1.3 and 2.3 (Mixed particle exposure). We have processed tissues collected in this study and find that BrdU positive cells are reduced in the iron + proton condition relative to control and iron alone conditions, 1 month after irradiation. Importantly, we did not detect any differences between male and female mice with regard to radiation effects on neurogenesis. Behavioral analyses at 6 and 12 months post-irradiation showed no difference between irradiated males and females (100 cGy protons). However we did observe a unique behavioral phenotype in male mice subjected to sequential irradiation with iron and protons: there was no decrease in freezing behavior with irradiation in the contextual task; however, mice irradiated with iron and protons showed a dramatic increase in freezing behavior in the novel context. Although we are not sure what this means, one interpretation is that the mice are hyper-vigilant, perhaps due to increased anxiety.

A third run at NSRL was conducted in May 2010, and involved 150 mice being irradiated with protons followed by delayed exposure to inhaled LPS (Aim 4). Mice were tested at 3 and 12 months post irradiation. Behavioral analyses reveal an effect of LPS, but no effect of radiation and no synergy between radiation and LPS exposure as anticipated. Histological evaluation of tissues for markers of neuroinflammation as well as effects on neurogenesis is currently underway. Additional analyses of blood and lung tissue from these mice as well as selected groups of mice irradiated in the first two runs is planned for the upcoming grant period.

Seventy-five AD transgenic mice (APPswe/PS-1d9; mixed male and female) were irradiated at 3 months of age using 100 cGy of 56Fe (1 GeV/n) particles during NSRL Run 11A. These mice will be behaviorally tested and sacrificed for tissue analysis at 9 months of age to determine the effects of space radiation on Alzheimer’s plaque pathology.

During NSRL 11A we were also able to expose 75 male wild-type mice to a modeled solar flare event using a range of proton energies and a total estimated dose of 200 cGy. These mice and 75 sham-exposed mice will be behaviorally tested and sacrificed at 3, 6 and 12 months post-irradiation.

A second series of exposures was conducted as part of NSRL Run 09C (November 17-20) and involved nearly 750 mice. These studies comprised experiments 1.2 and 2.2 (Sex Differences) as well as 1.3 and 2.3 (Mixed particle exposure). As mentioned in the previous progress report, this strategy conserved animal numbers since male animals with protons alone are common to both paradigms. We have collected tissues from this study through the 6 month time point, but will not be processing them for immunohistochemical staining or RNA analysis until the 12 month tissues are available. However, we did carry out fear-conditioning behavioral experiments with the 6 month group. Similar to the proton alone study, we did not see evidence of decreased hippocampal dependent learning in this paradigm following 100 cGy of protons, with or without 10 cGy of 56Fe HZE irradiation. We will be adding a second behavioral task for the 12 month time point.

A third run at NSRL was conducted in May 2010, and involved 150 mice irradiated with protons followed by delayed exposure to inhaled LPS (Aim 4). An initial set of mice has been sacrificed and tissues (brain, lung and blood) collected for a 3 month time point. Another set will be tested at 12 months. Tissues are currently being processed from this study.

Because of the late time points, specific conclusions arising from this study will have to wait. We anticipate completion of all analyses during year 2 of the award. With regard to additional studies, we are scheduled to participate in NSRL Run 09C, and will subject approximately 750 mice to proton and HZE exposures in early November 2009. In particular, we will be carrying out experiments 1.2 and 2.2 (Sex Differences) as well as 1.3 and 2.3 (Mixed particle exposure) during this run. This strategy helps conserve animal numbers since male animals with protons alone are common to both paradigms. Future anticipated runs include Specific Aim 4 (lung susceptibility) in Spring 2010 and the Alzheimer’s mouse study (Specific Aim 3) in Fall 2010. Our efforts to get these exposures completed relates to the long time course of each experiment (12 months) and the time it takes to complete our behavioral, molecular and histological analyses.