PRAXIS.

Usmani, Manal

Department of Radiation Oncology

Dr. Charles Limoli, Dr. Vipan Parihar (mentor)

The radiation fields in space define tangible risks to the health of astronauts, and significant work in rodent models has clearly shown a variety of exposure paradigms to compromise CNS functionality. Despite our current knowledge, sex differences regarding the risks of space radiation exposure on cognitive function remain poorly understood which is potentially problematic given that thirty percent of astronauts are women. While work from us and others have demonstrated pronounced cognitive decrements in male mice exposed to charged particle irradiation, here we show that female mice exhibit significant resistance to adverse neurocognitive effects of space radiation. Twelve weeks following 4He ion exposure irradiated male mice demonstrated significant deficits in object and place recognition memory. Additionally, we determined that as opposed to female cohorts, exposure to 4He ions caused a significant decline of dendritic spine density along hippocampal neurons in male mice. These data indicate that fundamental differences in inflammatory cascades between male and female mice may drive divergent CNS radiation responses that differentially impact the structural plasticity of neurons and neurocognitive outcomes following cosmic radiation exposure.

Astronauts are exposed to radiation in space that can have adverse effects on cognitive functioning. Galactic cosmic rays (GCR) contain 85% high energy protons, 14% helium ions, and other high energy nuclei (Nelson, 2016). The radiation fields in space have been measured and considerable information concerning the types, fluences and energies of charged particles that contribute to the expected doses astronauts would incur within and beyond the Earth's protective magnetosphere (Nelson, 2016). The measured dose rates and mission duration for a roundtrip mission to Mars are not expected to exceed 0.4-5 Gy, where the majority (~80%) of whole body exposures will be due to lighter particles (e.g. protons and helium ions) (Nelson, 2016).

Acute and chronic brain tissue alterations arise from the damaging effects of highly energetic charged particles that can traverse the tissues of the body (Cucinotta, 2014; Cucinotta and Cacao, 2019). Past work with mice has demonstrated that whole body exposure to charged particles can disrupt behavioral performance which can be linked to impairments in the hippocampus and medial prefrontal cortex (mPFC) (Britten et al., 2014; Parihar et al., 2015a; Parihar et al., 2016).

Neuronal synaptic areas are reshaped likely due to inflammation. This is hypothesized to occur through decrements in dendritic branches and spines thereby reducing the structural complexity of neurons throughout distinct regions of the brain which has numerous impacts on neurotransmission and cognition (Britten et al., 2020; Parihar et al., 2016). As the majority of such studies have been conducted exclusively with male mice, these results conclude that male rodent brains are vulnerable to exposures from 4He and other charged particle types (Parihar et al., 2018; Raber et al., 2018). There remains a gap for research on female rodents in this field. Current studies have found female mice to be more resistant than males to behavioral impairments and dendritic spine loss. This is still an active area of research, but a prominent hypothesis is that the complement cascade is implicated as an underlying cause (Hinkle et al., 2019). The high mobility group box 1 (HMGB1) is involved in pro-inflammatory signaling and increased levels are suggestive of the activation of these pathways. We will investigate this mechanistic pathway through levels of HMGBI in immunostaining analysis to establish what is happening at the molecular level leading to structural damage.

In this paper, we aimed to elucidate further potential sex-dependent differences in behavioral responses and the underlying mechanisms following cosmic radiation exposure. Our findings suggest that sex dependent differences in the response to cosmic irradiation can be linked to an attenuation of neuroinflammation and oxidative stress in female mice, providing a plausible explanation for their resistance to the adverse neurocognitive effects of 4He exposure.

MATERIALS AND METHODS

Mice Irradiation All experiments were carried out in accordance with the Institutional Animal Care and Use Committee at the University of California, Irvine, and were consistent with Federal guidelines. Obtained from the Jackson Lab, 6-month old male and female C57BL/6J mice were used in NOR behavior studies and 6-month old transgenic mice (strain TgThy1-EGFP) MJrsJ, Stock No. 007788) were utilized for structural analysis. Whole body irradiation of 5 cGy or 30 cGy was delivered at a rate of 5 cGy/min at the NASA Space and Radiation Laboratory (NSRL) at Brookhaven National Laboratory.

Behavioral testing Novel Object Recognition tested cognitive abilities which relies on a functioning medial prefrontal cortex. This test was administered post 3 day habituation for

10 min/day. Objects were chosen to be placed in the same spot for each trial and magnets were used to hold in place. The plastic material was kept constant, however shape, color, and size was different among the two toys for each test. The test day started off with 5 min of familiarization followed by 5 min rest in home cage while the toys were cleaned with 70% ethanol. Mice explored the objects for 5 min. The discrimination index was calculated through hand scoring data using a sensitive stop watch: Structural Assessment of Neurons to view dendritic structure it is imperative to study mice that have GFP in order to visualize and image at least 60 micron thick sections on the confocal microscope. The green fluorescence allows micromorphometric analysis where dendrites and spines can be rigorously distinguished. Thy1-EGFP mice were bred, genotyped, and perfused by 4% (wt/vol) paraformaldehyde 6 weeks post irradiation. Dissection was performed and brains were fixed in the same paraformaldehyde concentration. A cryostat (Leica) was used to section 100 μm thick brain sections and the Nikon Eclipse Ti C2 microscope subsequently provided images of the entire dendrite tree at 0.1 μm increments (Montay-Gruel et al.).

Statistical Analysis Statistical significance was assigned at p ≤ 0.05. Behavior and morphometric data were analyzing with two-way ANOVA with sex and radiation as the independent variables. GraphPad Prism v6.0 (GraphPad Software; La Jolla, CA) was used to analyze and create the figures in this paper.

RESULTS: For the same dose of energetic charged particles, it has been established that males exhibit significant impairments in the cortical and hippocampal regions while females show marked resistance to radiation. These conclusions were determined through the Novel Object Recognition (NOR) behavioral task. Six-month old male and female mice were tested 12-15 weeks post irradiation. Impairment is indicated through an inability to discriminate special, temporal, or object novelty. A discrimination index was calculated to measure preference or indifference for novelty object. A negative score is representative of indifference quantified by greater time exploring the familiar object whereas a positive score is representative of preference indicated through greater time exploring the novel object.

A two-way ANOVA showed that the total time spent exploring both objects was similar between irradiated and control groups. There was no evident effect of sex or radiation alone, with p = 0.10 and p = 0.10 respectively. No significant results for the sex and radiation test were observed either (p = 0.67). Both of these tests reveal that 4He exposure did not impair locomotor activity of the mice demonstrated by Fig. 1 A1-A2. A second ANOVA was conducted to investigate the control groups, concluding that both unirradiated male and female mice clearly preferred the novel object.

Fig. 1 B1 shows that there was a greater percentage of time spent exploring the novelty where males scored 63% (p < 0.0001) and females scored 60% (p < 0.0001). Irradiated female mice retained the ability to distinguish between the familiar and novel object (5cGy: 60%, p > 0.0001; 30cGy: 60 %, p > 0.0002, 60%; Fig. 1 B2), while males did not seem to prefer the novel object (5cGy: 45%, p > 0.99; 30cGy: p > 0.050, 45%; Fig. 1 B1).

Overall, Fig. 1 leads to the conclusion that post 12-week radiation, male recognition memory was impaired to a greater extent than females. An interaction effect between sex and radiation was demonstrated by a two-way ANOVA evaluating the discrimination index (DI), p = 0.48. Furthermore, a sex effect was evident (p < 0.0001) but a radiation effect was not significant (p = 0.10). A statistical group difference was highlighted through a marked reduction in time spent exploring novelty where control male mice produced a higher DI than irradiated males (5cGy: p = 0.0098; 30 cGy: p = 0.045; Fig. 1 C1). Females did not show results similar to males since the DI between control and irradiated counterparts was comparable (5cGy: p = 0.99, 30 cGy: p = 0.99; Fig. 1 C2).

Changes in high mobility group box 1 (HMGB1) protein were investigated following 4He irradiation in order to identify the inflammatory factors with the potential to influence neurocognitive outcomes. Immunohistochemical staining was performed to quantify HMGB1 levels in the hippocampus of both cohorts at 15 weeks following irradiation. Different levels of HMGB1 expression were detected between the control and irradiated mice in both cohorts. Two-way ANOVA demonstrated no significant radiation × sex interaction (F 1, 16 = 0.99, p = 0.33), no sex (F 1, 16 = 0.006, p = 0.93) effect but revealed a significant radiation effect (F 1, 16 = 9.26, p = 0.008).

In Figure 2, it can be seen that the absence of a significant overall sex effect between the groups precluded further post-hoc analysis. T-tests showed a 1.5 fold increase in the number of cells expressing HMGB1 in male mice following irradiation (p = 0.03, Fig. 2). Noteworthy again, the expression of HMGB1 in female mice was unaffected by irradiation. Despite the fact that the radiation enhances the number of HMGB1+ cells in male mice, we observed no sex effect on HMGB1 expression following irradiation.

Function is related to structure and these behavioral conclusions must have underlying structural alterations in the brain. Imaging analysis showed that 4He ion irradiation has a sex-specific effect on neuronal dendritic structure. Specifically, dendritic spine number and the molecular layer volume of the dentate gyrus were impacted (Fig. 3).

A significant radiation effect on spine number (p =0.006) was observed, which was not accompanied by a sex alone or sex and radiation interaction observation (p > 0.05). Fig. 3 A, C (5cGy) demonstrates that irradiated males have a lower spine number compared to their non-irradiated counterparts (p = 0.04). In contrast, females (5 cGy) have similar numbers of spines between control and treated groups with p = 0.81 seen through Fig. 3 B, C. Spine volume was reduced only for males and was not reduced significantly enough for female mice (Fig. 3 D, E, F), highlighting the correlation between behavior results and structural analysis results. The significant cognitive decrements in male NOR testing could be explained by decreased spine number and volume.

DISCUSSION: Together, results of NOR and structural data provide compelling evidence that male and female mice exhibit divergent responses following 4He ion low dose radiation exposure. Previous studies have shown that male rodents have decreased memory function post irradiation in NOR testing (Raber et al., 2014; Parihar et al., 2016; Raber et al., 2018). Additionally, data on dendritic morphology variables for small charged particle radiation such as 56Fe particles were suggested to have adverse effects on spine density and morphology (Allen et al., 2015). Low dose 48Ti particles have previously been seen to decrease dendritic complexity and spine density in the medial prefrontal cortex (Parihar et al., 2015; Parihar et al., 2016). These studies are consistent with our findings suggesting greater susceptibility to cognitive decrements in the hippocampal and cortical regions in male mice exposed to the same radiation and dose as females.

The HMGB1 results showed that there was a markedly increased in male mice 15 weeks following irradiation, a response that was not observed in female mice. HMGB1–mediated inflammatory pathways have been implicated in learning and memory deficits in CNS disorders, such as Alzheimer’s disease, Parkinson’s disease, temporal lobe epilepsy (Paudel et al., 2018; Webster et al., 2019). This data corroborates the behavior and structural analysis results, and provides an explanation in relation to the difference in immune response to radiation between the sexes.

Female radioresistance is likely due to estrogen signaling and its effects. It plays key neuroprotective roles in mitigation of cognitive deficits, mood disorders, and neurodegenerative disorders through anti-inflammatory properties (Behl and Manthey, 2000’ Norbury et al., 2003; Pratap et al., 2016). Recent studies showed that it may promote dendritic and axonal growth which can account for the radiation-induced reductions in dendritic morphology (Parihar et al., 2016; Lu et al., 2019). Future studies will focus on exploring the neuroprotective mechanisms that allow females to retain hippocampal function related to learning and memory abilities when faced with space-like radiation. Quantification of estrogen receptors in both sexes would potentially be an important parameter to investigate.

This study contributes to generating female mice data for effects of space-like irradiation on cognitive function, and confirms a sex-dependent disease onset. The implications of this research include developing countermeasures for space travel that inherently exposes the whole body to particle irradiation. Organizations such as NASA are invested in protecting and preserving the health of astronauts that want to accomplish a trip to Mars.

ACKNOWLEDGMENTS

I would like to thank Dr. Parihar for his constant mentorship on this project and Dr. Limoli for this research assistant opportunity.

Sources:

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