NOTE: End date changed to 03/31/2023 per NSSC information (Ed., 10/12/22)

Hypothesis: Exposure to a complex GCR mixture will decrease translationally-relevant cognitive function and negatively impact associated cellular and molecular processes in female and male mice, and CDDO-EA will mitigate these changes.

Aim 1. We will determine the effects of (1a) 33-particle GCR (33-GCR) on hippocampal dentate gyrus (DG) function (pattern separation) and prefrontal cortex (PFC) functions (cognitive flexibility, extinction of stimulus-response) in 6-month-old mature male C57BL/6J mice and (1b) CDDO-EA on 33-GCR-induced cognitive changes.

Aim 2. We will also define DG neurogenesis and DG and PFC inflammation in female and male mice exposed to 33-GCR or Sham with and without CDDO-EA.

Methods: Mature C57BL/6J male and female mice will receive CDDO-EA (400 mg/kg, n=80) or Control Chow (Control; n=80) for 5 days (3 days before, during, and 1 day after 33-GCR). Mice will receive either Sham or whole-body 33-GCR (~73% 1H, 18% 4He, 2.4% 12C, 3.1% 16O, 1.6% 28Si, 0.9% 48Ti, 0.8% 56Fe; 750mGy) with the 4 groups being Control/Sham, Control/33-GCR, CDDO-EA/Sham, CDDO-EA/33-GCR (n=16/group). To complement pilot data with female mice, in Aim 1, 2-mon post-irradiation male mice will undergo general touchscreen training, location discrimination (LD), and acquisition and extinction of stimulus-response. In Aim 2, brains will be collected from male and female mice at three timepoints post-33-GCR: 3 days, 2-mon, and 11-mon. 2- and 11-mon groups will be given bromodeoxyuridine (BrdU, i.p. 180 mg/kg) 2 hrs prior to perfusion and indices of neurogenesis and inflammation will be quantified via unbiased stereology. In the 3-day group, protein expression of Nrf2/ARE signaling targets will be quantified in the DG and PFC.

Aim 1 deliverables include measures of LD (session length, # trials, % correct, etc.) and acquisition and extinction (session length, # responses, # omissions, days to completion) in male mice, and the impact of 33-GCR vs. Sham and CDDO-EA vs. Control Chow on these measures.

Aim 2 deliverables include indices of DG neurogenesis (# of Ki67-, DCX-, BrdU-immunoreactive [+] cells) and DG and PFC inflammation (# of Iba-1+, Glial fibrillary acidic protein (GFAP) + cells)), and protein expression of signaling mediators downstream from nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE).

Significance: It is imperative to understand how a mission-relevant GCR mixture influences cognition, and determination of GCR-induced cellular and molecular changes enables identification of appropriate countermeasures. The proposed work in male and female mice will complement our already-completed cognitive study in female mice (manuscript in preparation). Given the similarity between cognitive function in mice and humans, the resulting data will allow more accurate estimation of space-radiation risks to both male and female astronauts.

Our lab has made progress on our NASA-funded research as described below:

NASA Human Exploration Research Opportunities (HERO) (PI: Yun)-funded publications (7):

1. Tran FH, Spears SL, Ahn KJ, Eisch AJ, Yun S. Does chronic systemic injection of the DREADD agonists clozapine-N-oxide or Compound 21 change behavior relevant to locomotion, exploration, anxiety, and depression in male non-DREADD-expressing mice? Neurosci Lett. 2020 Nov 20;739:135432.

2. Clark LR, Yun S, Acquah NK, Kumar PL, Metheny HE, Paixao RC, Cohen AS, Eisch AJ. Mild traumatic brain injury induces transient, sequential increases in proliferation, neuroblasts/immature neurons, and cell survival: A TimeCourse study in the male mouse dentate gyrus. Front Neurosci. 2021 Jan 7;14:612749.

3. Soler I, Yun S, Reynolds RP, Whoolery CW, Tran FH, Kumar PL, Rong Y, DeSalle MJ, Gibson AD, Stowe AM, Kiffer FC. Multi-domain touchscreen-based cognitive assessment of C57BL/6J female mice shows whole-body exposure to 56Fe particle space radiation in maturity improves discrimination learning yet impairs stimulus-response rule-based habit learning. Front Behav Neurosci. 2021 Oct 11;15:722780.

4. Kiffer FC, Luitel K, Tran FH, Patel RA, Guzman CS, Soler I, Xiao R, Shay JW, Yun S, Eisch AJ. Effects of a 33-ion sequential beam galactic cosmic ray analog on male mouse behavior and evaluation of CDDO-EA as a radiation countermeasure. Behav Brain Res. 2022 Feb 15;419:113677.

5. Cotter KM, Bancroft GL, Haas HA, Shi R, Clarkson AN, Croxall ME, Stowe AM, Yun S, Eisch AJ. Use of an automated mouse touchscreen platform for quantification of cognitive deficits after central nervous system injury. In: Karamyan, VT, Stowe, AM, eds. Neural Repair. Methods Mol Biol. Vol. 2616. New York, NY: Humana, 2023. p. 279-326.

6. Guez-Barber D, Colon LM, Raphael D, Wragan MA, Yun S, Eisch AJ. Female and male microglia are not different in the dentate gyrus of postnatal day 10 mice. Neurosci Lett. 2023 Apr 23;803:137171.

7. Yun S, Soler I, Tran FH, Haas HA, Shi R, Bancroft GL, Suarez M, de Santis CR, Reynolds RP, Eisch AJ. Behavioral pattern separation and cognitive flexibility are enhanced in a mouse model of increased lateral entorhinal cortex-dentate gyrus circuit activity. Front Behav Neurosci. 2023 Jun 1;17:1151877.

NASA HERO (PI Yun)-funded publications in prep (3):

1. Yun S, Kiffer FC, Guzman CS, Soler I, Bancroft GL, Haas HA, Shi R, Patel R, Lara-Jiménez J, Kumar PL, Tran FH, Ahn KJ, Rong Y, Luitel K, Shay JW, Eisch AJ. Contemporaneous ingestion of the antioxidant CDDO-EA prevents galactic cosmic ray-induced decline in simple stimulus-response learning and hippocampal neurogenesis in female C57BL/6J mice.

2. Kiffer FC, Hamandi A, Russo A, Johnson N, Roura GC, Gautreaux E, Bathwal M, Yang E, Gawon K, Kogawa L, Stanislaus D, Sil A, Chen A, Shi R, Gillis S, Barber DG, Yun S, Eisch AJ. Effects of a fractionated whole-body exposure to 56Fe high-energy particles on social behavior and neural activation in female and male C57BL/6J mice.

3. Yun S*, Mahajan A, Bancroft GL, Haas HA, Wallen-Friedman E, de Santis CR, Vasisht S, Takano H, Kiffer, FC, Eisch AJ. Exposure of Male and Female Mice to 33-Particle Galactic Cosmic Radiation (GCR): Impact on behavioral pattern separation and Pavlovian learning, and Prevention of GCR-induced Deficits by Acute Treatment with the Anti-inflammatory CDDO-EA *Corresponding author.

NASA HERO (PI: Yun) project-related findings:

We examined the impact of exposure to 33-particle galactic cosmic radiation (GCR) on cognitive performance in fully mature male and female mice and mitigation of 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO)-ethylamide (CDDO-EA), an antioxidant provided to the mice around the time of irradiation. We purchased male (n=124) and female (n=48) wildtype (C57BL/6J) mice for NSRL 22A (April 20, 2022). The variables for this experiment were irradiation (Sham and 33-GCR) and antioxidant (Veh or CDDO-EA), resulting in four groups of mice/sex (Sham/Veh, 33-GCR/Veh, Sham/CDDO-EA, 33-GCR/CDDO-EA; n=12/group/sex). After BNL, the mice were returned to CHOP. Female and male mice then were assessed for: monthly weight checks, home cage locomotion activity monitoring (18hrs), spontaneous location recognition test (a pattern separation test), training and testing on an automated touchscreen platform (Pavlovian learning) and elevated plus maze (a test for anxiety). In short, we found the acute exposure to 33-GCR (0.75 Gy, 1hr 40min) at 6 mon-old increases behavioral pattern separation– a type of high-resolution memory for discriminating similar stimuli– in male mice but not female mice. Also, administration of an antioxidant, CDDO-EA (4 mg/kg i.p for 3 days: the day before, on the day, and a day after IRR) normalizes male behavioral pattern separation. This test is highly sensitive to the dentate gyrus–a hippocampal subregion. Dentate gyrus (DG) activity was measured during the behavioral pattern separation via an indirect measurement of DG granular cell activity. We found that exposure to the 33-GCR decreases DG cell activity during exploration in similar stimuli which implies neural mechanisms of 33-GCR-induced behavioral pattern separation changes. Also, we tested association learning, a type of Pavlovian learning. We found that 33-GCR alone transiently decreases “sign tracking” behavior–approaches to the reward-related condition stimuli during Acq. in males, not females. CDDO-EA itself (in Sham, non-IRR mice) does not change “sign tracking” during Acq. in both males and females. Unexpectedly, 33-GCR exposure increased reversal learning during reverse learning to the acquired learning in male mice. However, female approaches to CS+ are unchanged during reversal learning.

There are several implications of our study. 1) The exposure to a mission-relevant dose of 33-GCR may increase pattern separation ability in male astronauts, but not female astronauts after a mission. 2) CDDO-EA ameliorates cognitive deficits after a mission. These rodent findings warrant additional study with radiation parameters that decrease cognition. 3) Exposure to a mission-relevant dose of GCR has a sex-specific influence on different forms of discrimination and associative learning. Thus, more work with 33-GCR is warranted. For example, as our study of real-time measurement of the DG activity in freely behaving animals gives insight into cellular mechanisms underlying 33-GCR-induced improvement of behavioral pattern separation, fMRI study in astronauts during or after their mission will be warranted. So far, we have shared these data each year at the NASA Human Research Program Investigators' Workshop (HRP IWS) meeting and Radiation Research in order to receive feedback on our approach and interpretations. The resulting data are the focus of several manuscripts we have in preparation/revision, and Dr. Yun (PI of this HERO grant) is first and/or senior author on each manuscript.

NOTE: End date changed to 03/31/2023 per NSSC information (Ed., 10/12/22)

Hypothesis: Exposure to a complex GCR mixture will decrease translationally-relevant cognitive function and negatively impact associated cellular and molecular processes in female and male mice, and CDDO-EA will mitigate these changes.

Aim 1. We will determine the effects of (1a) 33-particle GCR (33-GCR) on hippocampal dentate gyrus (DG) function (pattern separation) and prefrontal cortex (PFC) functions (cognitive flexibility, extinction of stimulus-response) in 6-month-old mature male C57BL/6J mice and (1b) CDDO-EA on 33-GCR-induced cognitive changes.

Aim 2. We will also define DG neurogenesis and DG and PFC inflammation in female and male mice exposed to 33-GCR or Sham with and without CDDO-EA.

Methods: Mature C57BL/6J male and female mice will receive CDDO-EA (400 mg/kg, n=80) or Control Chow (Control; n=80) for 5 days (3 days before, during, and 1 day after 33-GCR). Mice will receive either Sham or whole-body 33-GCR (~73% 1H, 18% 4He, 2.4% 12C, 3.1% 16O, 1.6% 28Si, 0.9% 48Ti, 0.8% 56Fe; 750mGy) with the 4 groups being Control/Sham, Control/33-GCR, CDDO-EA/Sham, CDDO-EA/33-GCR (n=16/group). To complement pilot data with female mice, in Aim 1, 2-mon post-irradiation male mice will undergo general touchscreen training, location discrimination (LD), and acquisition and extinction of stimulus-response. In Aim 2, brains will be collected from male and female mice at three timepoints post-33-GCR: 3 days, 2-mon, and 11-mon. 2- and 11-mon groups will be given bromodeoxyuridine (BrdU, i.p. 180 mg/kg) 2 hrs prior to perfusion and indices of neurogenesis and inflammation will be quantified via unbiased stereology. In the 3-day group, protein expression of Nrf2/ARE signaling targets will be quantified in the DG and PFC.

Aim 1 deliverables include measures of LD (session length, # trials, % correct, etc.) and acquisition and extinction (session length, # responses, # omissions, days to completion) in male mice, and the impact of 33-GCR vs. Sham and CDDO-EA vs. Control Chow on these measures.

Aim 2 deliverables include indices of DG neurogenesis (# of Ki67-, DCX-, BrdU-immunoreactive [+] cells) and DG and PFC inflammation (# of Iba-1+, Glial fibrillary acidic protein (GFAP) + cells)), and protein expression of signaling mediators downstream from nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE).

Significance: It is imperative to understand how a mission-relevant GCR mixture influences cognition, and determination of GCR-induced cellular and molecular changes enables identification of appropriate countermeasures. The proposed work in male and female mice will complement our already-completed cognitive study in female mice (manuscript in preparation). Given the similarity between cognitive function in mice and humans, the resulting data will allow more accurate estimation of space-radiation risks to both male and female astronauts.

Our lab has made progress on our NASA-funded research as described below: NASA Human Exploration Research Opportunities (HERO) (PI: Yun)-funded publications (4):

Tran FH, Spears SL, Ahn KJ, Eisch AJ, Yun S. Does chronic systemic injection of the DREADD agonists clozapine-N-oxide or Compound 21 change behavior relevant to locomotion, exploration, anxiety, and depression in male non-DREADD-expressing mice? Neurosci Lett. Elsevier. 2020 Nov.

Clark LR, Yun S, Acquah NK, Kumar PL, Metheny HE, Paixao RCC, Cohen AS, Eisch AJ. Mild traumatic brain injury induces transient, sequential increases in proliferation, neuroblasts/immature neurons, and cell survival: A TimeCourse study in the male mouse dentate gyrus. Front Neurosci. 2020 Dec.

Soler I*, Yun S*+, Reynolds RP, Whoolery CW, Tran FH, Kumar PL, Rong Y, DeSalle MJ, Gibson AD, Stowe AM, Kiffer FC, Eisch AJ+. *Co-first authors, +Co-corresponding authors. Multi-domain touchscreen-based cognitive assessment of C57BL/6J female mice shows whole-body exposure to 56Fe particle space radiation in maturity improves discrimination learning yet impairs stimulus-response rule-based habit learning. Front Behav Neurosci. 2021 Oct 11;15:722780. PMCID: PMC8543003.

Kiffer FC, Luitel K, Tran FH, Patel RA, Guzman CS, Soler I, Xiao R, Shay JW, Yun S, Eisch AJ. Effects of a 33-ion sequential beam galactic cosmic ray analog on male mouse behavior and evaluation of CDDO-EA as a radiation countermeasure. Behav Brain Res. 2022;419:113677.

NASA HERO (PI Yun) additional project-related progress:

We examined the impact of exposure to 33-particle galactic cosmic radiation (GCR) on cognitive performance in fully mature male and female mice and the mitigation of 2-cyano-3,12-dioxoolean-1,9(11)-dien-28-oic acid (CDDO), an antioxidant provided to the mice around the time of irradiation. We purchased male (n=124) and female (n=48) wildtype (C57BL/6J) mice for NSRL 22A (April 20, 2022). The variables for this experiment were irradiation (Sham and 33-GCR) and antioxidant (Veh or CDDO-EA), resulting in four groups of mice/sex (Sham/Veh, 33-GCR/Veh, Sham/CDDO-EA, 33-GCR/CDDO-EA; n=12/group/sex). After BNL, the mice were returned to CHOP. Female and male mice were then assessed for: monthly weight checks, home cage locomotion activity monitoring (18hrs), spontaneous location recognition test (a pattern separation test), training and testing on an automated touchscreen platform (Pavlovian learning) and elevated plus maze (a test for anxiety). We are currently analyzing all behavior data, including performance on the pattern separation test, touchscreen-based Pavlovian learning tests, and anxiety tests. Brains from all mice were collected and will be processed for dependent measures based on the behavioral results. Also, we run a spontaneous location recognition test (a pattern separation test) with fiber photometry recording to assess the impact of 33-GCR and CDDO on the dentate gyrus (DG) granular cell activity in behaving animals and currently analyze the data.

We have shared these data each year at NASA Human Research Program (HRP) and Radiation Research in order to receive feedback on our approach and interpretations. The resulting data are the focus of several manuscripts we have in preparation/revision, and Dr. Yun (PI of this HERO grant) is first and/or senior author on each manuscript.

Hypothesis: Exposure to a complex GCR mixture will decrease translationally-relevant cognitive function and negatively impact associated cellular and molecular processes in female and male mice, and CDDO-EA will mitigate these changes.

Aim 1. We will determine the effects of (1a) 33-particle GCR (33-GCR) on hippocampal dentate gyrus (DG) function (pattern separation) and prefrontal cortex (PFC) functions (cognitive flexibility, extinction of stimulus-response) in 6-month-old mature male C57BL/6J mice and (1b) CDDO-EA on 33-GCR-induced cognitive changes.

Aim 2. We will also define DG neurogenesis and DG and PFC inflammation in female and male mice exposed to 33-GCR or Sham with and without CDDO-EA.

Methods: Mature C57BL/6J male and female mice will receive CDDO-EA (400 mg/kg, n=80) or Control Chow (Control; n=80) for 5 days (3 days before, during, and 1 day after 33-GCR). Mice will receive either Sham or whole-body 33-GCR (~73% 1H, 18% 4He, 2.4% 12C, 3.1% 16O, 1.6% 28Si, 0.9% 48Ti, 0.8% 56Fe; 750mGy) with the 4 groups being Control/Sham, Control/33-GCR, CDDO-EA/Sham, CDDO-EA/33-GCR (n=16/group). To complement pilot data with female mice, in Aim 1, 2-mon post-irradiation male mice will undergo general touchscreen training, location discrimination (LD), and acquisition and extinction of stimulus-response. In Aim 2, brains will be collected from male and female mice at three timepoints post-33-GCR: 3 days, 2-mon, and 11-mon. 2- and 11-mon groups will be given bromodeoxyuridine (BrdU, i.p. 180 mg/kg) 2 hrs prior to perfusion and indices of neurogenesis and inflammation will be quantified via unbiased stereology. In the 3-day group, protein expression of Nrf2/ARE signaling targets will be quantified in the DG and PFC.

Aim 1 deliverables include measures of LD (session length, # trials, % correct, etc.) and acquisition and extinction (session length, # responses, # omissions, days to completion) in male mice, and the impact of 33-GCR vs. Sham and CDDO-EA vs. Control Chow on these measures.

Aim 2 deliverables include indices of DG neurogenesis (# of Ki67-, DCX-, BrdU-immunoreactive [+] cells) and DG and PFC inflammation (# of Iba-1+, Glial fibrillary acidic protein (GFAP) + cells)), and protein expression of signaling mediators downstream from nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE).

Significance: It is imperative to understand how a mission-relevant GCR mixture influences cognition, and determination of GCR-induced cellular and molecular changes enables identification of appropriate countermeasures. The proposed work in male and female mice will complement our already-completed cognitive study in female mice (manuscript in preparation). Given the similarity between cognitive function in mice and humans, the resulting data will allow more accurate estimation of space-radiation risks to both male and female astronauts.