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Project Title:  Role of Oxidative Stress in Mediating the Effects of Combined Exposure to Simulated Microgravity and Radiation on Neurovascular Remodeling in Mouse Reduce
Fiscal Year: FY 2019 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Animal Biology: Vertebrate  
Start Date: 10/01/2013  
End Date: 09/04/2019  
Task Last Updated: 09/10/2018 
Download report in PDF pdf
Principal Investigator/Affiliation:   Mao, Xiao Wen  M.D. / Loma Linda University 
Address:  Radiation Medicine 
11175 Campus Street, Rm A1010, Chan Shun Pavilion 
Loma Linda , CA 92350-0001 
Email: xmao@llu.edu 
Phone: 909-558-8373  
Congressional District: 31 
Web:  
Organization Type: UNIVERSITY 
Organization Name: Loma Linda University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Gridley, Daila  Ph.D. Loma Linda University 
Hartman, Richard  Ph.D. Loma Linda University 
Pecaut, Michael  Ph.D. Loma Linda University 
Key Personnel Changes / Previous PI: July 2016 report: No changes.
Project Information: Grant/Contract No. 80NSSC17K0693 ; NNX13AL97G 
Responsible Center: NASA ARC 
Grant Monitor: Sato, Kevin  
Center Contact: 650-604-1104 
kevin.y.sato@nasa.gov 
Solicitation / Funding Source: 2012 Space Biology NNH12ZTT001N 
Grant/Contract No.: 80NSSC17K0693 ; NNX13AL97G 
Project Type: GROUND 
Flight Program:  
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Space Biology Element: (1) Animal Biology: Vertebrate
Space Biology Cross-Element Discipline: (1) Neurobiology
Space Biology Special Category: (1) Translational (Countermeasure) Potential
Flight Assignment/Project Notes: NOTE: Extended to 9/4/2019 per F. Hernandez/ARC (Ed., 9/10/18)

NOTE change in grant number and end date per NSSC information and F. Hernandez/ARC (Ed., 1/18/2018)

NOTE: End date changed to 8/31/2018 per F. Hernandez/ARC (Ed., 6/27/17)

NOTE: End date changed to 9/30/2017 per NSSC (Ed., 7/4/16)

Task Description: One of the main concerns for long-term deep manned space missions are health risks associated with altered gravitational environment and prolonged exposure to low-dose radiation above levels normally found on Earth. Microgravity and radiation exposure have been known to produce a number of neurological disturbances and neurodegeneration by space flight condition. However, the pathophysiological process from adaptive response to irreversible oxidative damage in the brain vasculature and the underlying mechanism(s) of these disturbances are less studied and remain unclear.

Our proposal seeks to fill in the gap by testing the hypothesis that NADPH oxidase is a critical source of the neurovascular oxidative stress following space flight conditions that mediates vascular remodeling in the brain, thus disrupting communication between endothelial cells and astrocytes and altering production of extracellular matrix (ECM) proteins. It is further proposed that these changes will contribute to increased vascular permeability and blood-brain barrier (BBB) disturbance, thus resulting in neurological deficit.

Our specific aims are 1) Define the causal relationships between space flight condition induced NADPH oxidase expression, vascular damage, and BBB function following microgravity and/or low-dose irradiation in mature mice using neuropathology, stereological, and automated image analysis, and neurobehavioral outcomes. 2) Determine if space flight condition-induced oxidative stress is mediated through NADPH oxidase in brain microvasculature.

Nox2 (a subunit of NADPH oxidase) gene knockout (Nox2(-/-)) mice, and wild-type (Nox2(+/+)) C57BL/6 mice will be used in this ground-based animal study. Hindlimb unloading (HLU) will be used to model the unloading, fluid shift, and physiological stress aspects of the microgravity component. Low-dose/low-dose-rate (LDR) gamma-irradiation (0.5 Gy at 0.01 cGy/h) will be delivered to the whole-body of mature adult mice to simulate the radiation component for over 21 days while the animals are tail-unloaded in cages for microgravity simulation. We will evaluate the radiation- and microgravity-induced brain vascular and tissue remodeling at multiple time points (1 day to 12 months post-irradiation).

Together, our unique, integrative, and quantitative activities with advanced imaging techniques, stereological analysis, and behavioral tests will provide insight into the molecular mechanisms of space flight condition-induced oxidative damage on brain tissue and vascular remodeling. Understanding how factors and environmental stress impact on vasculature, tissue remodeling, and function will increase our knowledge and focus toward more effective countermeasures during human space flight and planetary exploration. Our study will also lend new insights into the causes and possible treatments of debilitating neurovascular-related disease and neurodegeneration by targeting NADPH oxidase activation.

Research Impact/Earth Benefits: Oxidative stress in central nervous system (CNS) is a major contributor to brain injury and aging. There are strong indications that the physiological effects of space flight are similar to those seen in some neurodegenerative diseases and aging: multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease. Our study will provide the first detailed description of combined effects of microgravity and LDR radiation on oxidative stress-induced brain tissue and microvessel network remodeling and underlying mechanism(s) of potential interaction of space flight environmental components over a 12-month observation period. Our research will provide important input to elucidate cellular pathways of response and adaptation to stress imposed by environmental conditions in the brain vasculature. Understanding how factors and environmental insults impact on vasculature and tissue remodeling and function will increase our knowledge and help focus the approach toward more effective countermeasures during human space flight and planetary exploration. Our study might also lend new insights into the causes and possible treatments of debilitating neurovascular-related diseases and neurodegeneration.

Task Progress & Bibliography Information FY2019 
Task Progress: FINAL REPORTING SEPTEMBER 2019

All the experiments and data analysis have been completed as planned. So far, we have finished four batches of wild-type (WT) animal studies for specific aim 1, at 7 days, 1, 3, and 9 month time points and Nox2 knockout mice study as proposed in specific aims 1 and 2. We have examined protein markers associated with the induction of oxidative stress and apoptosis in the brain using immunostaining, metabolic response using metabolic biochemical assays, hematological parameters, and behavioral responses after combined exposure to simulated microgravity and low-dose radiation. Our data provide the first evidence that prolonged exposure to simulated microgravity and LDR radiation is associated with increased oxidative stress biomarkers which may increase the likelihood of brain injury and reduced antioxidant defense. NOX2-containing nicotinamide adenosine dinucleotide phosphate (NADPH oxidase) may contribute to spaceflight environment-induced oxidative stress. These results suggest that microgravity may lead to changes in exploratory/risk-taking behaviors in the absence of other sensorimotor or cognitive deficits and that combined microgravity and a chronic, low dose of gamma radiation may lead to blood-brain barrier dysfunction. These results have been published in four peer reviewed journals: Radiation Research, Nature npj Microgravity, PLOS One.

We have received supplement funding in October, 2017 to use"omics"-based molecular phenotyping approach for identification and characterization of genomic signatures in eye and brain associated with low-dose radiation and simulated microgravity at 1-and 4- month time points. Omics analyses were performed for full genome transcriptomics including RNA-Seq HT Ribo-depletion library preps and RNA spike-control. DNA methylation of reduced representation bisulfite sequencing (RRBS). The dataset has been submitted to NASA GeneLab site, through NASA IT system. One manuscript titled: Mice Exposed to Combined Chronic Low-Dose Irradiation and Modeled Microgravity Develop Long-Term Neurological Sequelae has been published in International Journal of Medical Sciences (IJMS) to evaluate simulated radiation and microgravity induced changes in the brain related to cellular structure, oxidative stress, immune response, and metabolic function.

ANNUAL REPORTING SEPTEMBER 2018: The purpose of this study was to determine whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived stress can account for unloading- and radiation-induced endothelial damage and neurovascular remodeling in a mouse model. Wild-type (WT, Nox2 (+/+)) C57BL/6 mice or Nox2 (-/-) (B6. 129S6-CYBBM) knockout (KO) mice were placed into one of the following groups: age-matched control, hindlimb unloading (HLU), low-dose/low-dose-rate radiation (LDR), or HLU+LDR simultaneously for 21 days, and were then sacrificed 1 month later. Anti-orthostatic tail suspension was used to model the unloading, fluid shift, and physiological stress aspects of microgravity. The LDR was delivered using 57Co plates (0.04Gy at 0.01cGy/h) to the whole body in order to simulate the radiation experienced while in space. Brains were isolated for characterization of various oxidative stress markers and vascular topology.

We received supplement funding in October, 2017 to use “omics”-based molecular phenotyping approach for identification and characterization of genomic signatures in eye and brain associated with low-dose radiation and simulated microgravity at 1- and 4– month time points. Omics analyses performed were

• RNAseq for full genome transcriptomics including RNA-Seq HT Ribo-depletion library preps and RNA spike-control.

• DNA methylation of reduced representation bisulfite sequencing (RRBS).

The assay has completed and dataset are in the process of submitting to NASA GeneLab site, through NASA IT system. Many canonical pathways were significantly activated/inhibited in the brain after HLU, LDgammaR, or the combination of both. Most robust changes are observed in LDgammaR + HLU group compared to control. At 1 months, the combination group induced significant activations of EIF2 phosphorylation signaling pathway in the brain, responsible for chronic neurodegeneration. Cellular immune response pathways including leukocyte extravasation signaling, as well as pathways responsible for cell growth, cell repair, and metabolic stress were also significantly altered compared to controls. The mRNA expression for many genes regulating oxidative stress (e.g., GNRH1, UCN3), extracellular matrix remodeling (e.g., Cldn3, FBLN1), endothelial cell biology (e.g., AQP1), and cognitive function (e.g., OXT, AVP) were significantly changed (p<0.05) after HLU+ LDgammaR compared to control. Gene analysis of retina tissues also showed that many key pathways responsible for photoreceptor function, oxidative stress, and metabolic function were significantly different between LDgammaR + HLU and control (p<0.05).

Bibliography Type: Description: (Last Updated: 09/27/2021) 

Show Cumulative Bibliography Listing
 
Abstracts for Journals and Proceedings Mao XW, Pecaut MJ, Hartman R, Nishiyama N, Bellone J, Boerma M, Nelson G. "Effects of simulated microgravity and/or low-dose radiation on neurovascular remodeling in the mouse brain and eye." Presented at 42nd Committee on Space Research (COSPAR) 2018 42nd Scientific Assembly, Pasadena, CA, July 14-22, 2018.

2nd Committee on Space Research (COSPAR) 2018 42nd Scientific Assembly, Pasadena, CA, July 14-22, 2018. , Jul-2018

Abstracts for Journals and Proceedings Mao XW, Nelson G, Jones T, Campbell-Beacher M, Stanbouly S, Nishiyama N, Rodriguez D, Ortloff L, Sun S, Sridharan V, Boerma M, Hauer-Jensen M. "Cardiac and vascular effects of space radiation - CSRR acute risk studies." 2018 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 22-25, 2018.

2018 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 22-25, 2018. , Jan-2018

Abstracts for Journals and Proceedings Mao XW. "Effects of Low-dose Radiation on Neurovascular Remodeling." Presented at the The Conference on Normal Tissue Radiation Effects and Countermeasure (CONTREC), Morrilton, AR, May 14-17, 2018.

The Conference on Normal Tissue Radiation Effects and Countermeasure (CONTREC), Morrilton, AR, May 14-17, 2018. , May-2018

Abstracts for Journals and Proceedings Mao XW. "Spaceflight and radiation induced microvessel and structural damage in ocular tissue." International Space Station Microgravity Space Medicine Meeting, American Institute of Aeronautics &Astronautics (AIAA), Manhattan Beach, CA, March 20, 2018.

International Space Station Microgravity Space Medicine Meeting, American Institute of Aeronautics &Astronautics (AIAA), Manhattan Beach, CA, March 20, 2018. , Mar-2018

Abstracts for Journals and Proceedings Mao XW, Boerma M, Nelson G, Shiba D, Shirakawa M, Takahash S, Delp M. "Impact of space flight or simulated microgravity combined with space radiation exposure on retinal oxidative damage." Presented at International Society for Gravitational Physiology (ISGP), Nagoya, Japan, May 26-June 1, 2019.

Abstracts. International Society for Gravitational Physiology (ISGP), Nagoya, Japan, May 26-June 1, 2019. , May-2019

Articles in Peer-reviewed Journals Mao XW, Nishiyama N, Campbell-Beachler M, Gifford P, Haynes KE, Gridley DS, Pecaut M. "Role of NADPH oxidase as a mediator of oxidative damage in low-dose radiated and hindlimb-unloaded mice." Radiation Research. 2017 Oct;188(4):392-9. Epub 2017 Aug 1. https://doi.org/10.1667/RR14754.1 ; PubMed PMID: 28763287 , Oct-2017
Articles in Peer-reviewed Journals Overbey EG, Paul AM, da Silveira WA, Tahimic CGT, Reinsch SS, Szewczyk N, Stanbouly S, Wang C, Galazka JM, Mao XW. "Mice exposed/ to combined chronic low-dose irradiation and modeled microgravity develop long-term neurological sequelae." Int J Mol Sci. 2019 Aug 22;20(17):E4094. https://doi.org/10.3390/ijms20174094 ; PubMed PMID: 31443374 , Aug-2019
Project Title:  Role of Oxidative Stress in Mediating the Effects of Combined Exposure to Simulated Microgravity and Radiation on Neurovascular Remodeling in Mouse Reduce
Fiscal Year: FY 2018 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Animal Biology: Vertebrate  
Start Date: 10/01/2013  
End Date: 09/04/2019  
Task Last Updated: 07/25/2017 
Download report in PDF pdf
Principal Investigator/Affiliation:   Mao, Xiao Wen  M.D. / Loma Linda University 
Address:  Radiation Medicine 
11175 Campus Street, Rm A1010, Chan Shun Pavilion 
Loma Linda , CA 92350-0001 
Email: xmao@llu.edu 
Phone: 909-558-8373  
Congressional District: 31 
Web:  
Organization Type: UNIVERSITY 
Organization Name: Loma Linda University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Gridley, Daila  Ph.D. Loma Linda University 
Hartman, Richard  Ph.D. Loma Linda University 
Pecaut, Michael  Ph.D. Loma Linda University 
Key Personnel Changes / Previous PI: July 2016 report: No changes.
Project Information: Grant/Contract No. 80NSSC17K0693 ; NNX13AL97G 
Responsible Center: NASA ARC 
Grant Monitor: Sato, Kevin  
Center Contact: 650-604-1104 
kevin.y.sato@nasa.gov 
Solicitation / Funding Source: 2012 Space Biology NNH12ZTT001N 
Grant/Contract No.: 80NSSC17K0693 ; NNX13AL97G 
Project Type: GROUND 
Flight Program:  
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Space Biology Element: (1) Animal Biology: Vertebrate
Space Biology Cross-Element Discipline: (1) Neurobiology
Space Biology Special Category: (1) Translational (Countermeasure) Potential
Flight Assignment/Project Notes: NOTE: Extended to 9/4/2019 per F. Hernandez/ARC (Ed., 9/10/18)

NOTE change in grant number and end date per NSSC information and F. Hernandez/ARC (Ed., 1/18/2018)

NOTE: End date changed to 8/31/2018 per F. Hernandez/ARC (Ed., 6/27/17)

NOTE: End date changed to 9/30/2017 per NSSC (Ed., 7/4/16)

Task Description: One of the main concerns for long-term deep manned space missions are health risks associated with altered gravitational environment and prolonged exposure to low-dose radiation above levels normally found on Earth. Microgravity and radiation exposure have been known to produce a number of neurological disturbances and neurodegeneration by space flight condition. However, the pathophysiological process from adaptive response to irreversible oxidative damage in the brain vasculature and the underlying mechanism(s) of these disturbances are less studied and remain unclear.

Our proposal seeks to fill in the gap by testing the hypothesis that NADPH oxidase is a critical source of the neurovascular oxidative stress following space flight conditions that mediates vascular remodeling in the brain, thus disrupting communication between endothelial cells and astrocytes and altering production of extracellular matrix (ECM) proteins. It is further proposed that these changes will contribute to increased vascular permeability and blood-brain barrier (BBB) disturbance, thus resulting in neurological deficit.

Our specific aims are 1) Define the causal relationships between space flight condition induced NADPH oxidase expression, vascular damage, and BBB function following microgravity and/or low-dose irradiation in mature mice using neuropathology, stereological, and automated image analysis, and neurobehavioral outcomes. 2) Determine if space flight condition-induced oxidative stress is mediated through NADPH oxidase in brain microvasculature.

Nox2 (a subunit of NADPH oxidase) gene knockout (Nox2(-/-)) mice, and wild-type (Nox2(+/+)) C57BL/6 mice will be used in this ground-based animal study. Hindlimb unloading (HLU) will be used to model the unloading, fluid shift, and physiological stress aspects of the microgravity component. Low-dose/low-dose-rate (LDR) gamma-irradiation (0.5 Gy at 0.01 cGy/h) will be delivered to the whole-body of mature adult mice to simulate the radiation component for over 21 days while the animals are tail-unloaded in cages for microgravity simulation. We will evaluate the radiation- and microgravity-induced brain vascular and tissue remodeling at multiple time points (1 day to 12 months post-irradiation).

Together, our unique, integrative, and quantitative activities with advanced imaging techniques, stereological analysis, and behavioral tests will provide insight into the molecular mechanisms of space flight condition-induced oxidative damage on brain tissue and vascular remodeling. Understanding how factors and environmental stress impact on vasculature, tissue remodeling, and function will increase our knowledge and focus toward more effective countermeasures during human space flight and planetary exploration. Our study will also lend new insights into the causes and possible treatments of debilitating neurovascular-related disease and neurodegeneration by targeting NADPH oxidase activation.

Research Impact/Earth Benefits: Oxidative stress in central nervous system (CNS) is a major contributor to brain injury and aging. There are strong indications that the physiological effects of space flight are similar to those seen in some neurodegenerative diseases and aging: multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease. Our study will provide the first detailed description of combined effects of microgravity and LDR radiation on oxidative stress-induced brain tissue and microvessel network remodeling and underlying mechanism(s) of potential interaction of space flight environmental components over a 12-month observation period. Our research will provide important input to elucidate cellular pathways of response and adaptation to stress imposed by environmental conditions in the brain vasculature. Understanding how factors and environmental insults impact on vasculature and tissue remodeling and function will increase our knowledge and help focus the approach toward more effective countermeasures during human space flight and planetary exploration. Our study might also lend new insights into the causes and possible treatments of debilitating neurovascular-related diseases and neurodegeneration.

Task Progress & Bibliography Information FY2018 
Task Progress: The purpose of this study was to determine whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived stress can account for unloading- and radiation-induced endothelial damage and neurovascular remodeling in a mouse model. Wild-type (WT, Nox2 (+/+)) C57BL/6 mice or Nox2 (-/-) (B6. 129S6-CYBBM) knockout (KO) mice were placed into one of the following groups: age-matched control, hindlimb unloading (HLU), low-dose/low-dose-rate radiation (LDR), or HLU+LDR simultaneously for 21 days, and were then sacrificed 1 month later. Anti-orthostatic tail suspension was used to model the unloading, fluid shift, and physiological stress aspects of microgravity. The LDR was delivered using 57Co plates (0.04Gy at 0.01cGy/h) to the whole body in order to simulate the radiation experienced while in space. Brains were isolated for characterization of various oxidative stress markers and vascular topology. Level of 4-hydroxynonenal (4-HNE) protein, a specific marker for lipid peroxidation, was measured. Expression of aquaporin-4 (AQP4), a water channel protein expressed in astrocyte endfeet, was quantified. Thirty days after simulated spaceflight, KO mice showed decreased apoptosis (p<0.05) in the brain compared to WT counterparts. The HLU-dependent increase in apoptosis in WT mice was not observed in KO mice. Level of 4-HNE protein was significantly elevated in the hippocampus of LDR+HLU group compared to controls in the WT mice (p<0.05). However, there was no significant differences among groups of Nox2-KO mice at 1-month time point. In contrast to findings in WT animals, superoxide dismutase (SOD) level and expression of AQP4 were similar among all KO groups. In summary, for most of the parameters, the oxidative response to HLU and LDR was suppressed in Nox2-KO mice. This suggests that Nox2-containing NADPH oxidase may contribute to space flight environment–induced oxidative stress.

To compare changes in leukocyte parameters in wild-type (WT) and Nox2(-/-) knockout (KO) mice 30 and 120 days after using a ground-based model for space flight. Six-month-old female Nox2(+/+) and Nox2(-/-) C57BL/6 mice (n=4-6/group) were exposed to whole-body low-dose/low-dose-rate (LDR) gamma-irradiation using 57Co plates (0.04 Gy at 0.01 cGy/h) and/or hindlimb unloaded (HLU) for 21 days. Mice were sacrificed with 100% CO2 at 30 or 120 days after the simulated space flight period and blood was collected via cardiac puncture. An automated ABC Vet Hematology Analyzer was used to obtain white blood cell (WBC), lymphocyte (LYM), monocyte (MON), and granulocyte (GRA) counts and percentages.

Thirty days after simulated space flight, KO mice showed increased GRA counts (P<0.005) and decreased LYM (P<0.001) and MON (P<0.05) counts compared to WT controls. This resulted in a shift away from %LYM (P<0.001) and %MON (P<0.001) towards %GRA (P<0.001). Similarly, hindlimb unloading caused increases in WBC (P<0.05) and GRA counts (P<0.05), leading to proportional shifts away from %LYM (P=0.005) and %MON (P<0.05) towards %GRA (P<0.005). Interestingly, the HLU-dependent increase in GRA was augmented in the KO mice, resulting in a KO x HLU interaction (P<0.05). Furthermore, unloading increased %GRA and decreased %MON, but only in the KO mice, leading to significant KO x HLU interactions (Ps<0.05). Although %LYM were generally lower in KO mice, the LDR-dependent decrease in this parameter noted in WT was reversed in KO mice. This led to a significant KO x LDR interaction (P<0.05).

At 120 days after simulated-space flight, there were several significant main effects of KO due to increases in WBC (P<0.001), MON (P<0.001), and GRA (P<0.001) counts. Because the KO-dependent increases in GRA counts was much higher than in other populations, this led to a shift away from %LYM (P<0.001) and %MON (P<0.001) towards %GRA (P<0.001). In contrast to the response at 30 days, the only significant main effect of HLU at 120 days was a decrease in %GRA (P<0.05). However, the HLU-dependent increase on MON counts noted in WT mice was amplified in KO mice. The HLU-dependent decrease in LYM counts was reversed in KO mice. This led to significant KO x HLU interactions (Ps<0.05) in both of these parameters. Finally, the LDR-dependent increase in %MON noted in WT mice was not observed in KO mice, leading to a significant KO x LDR interaction (P<0.05).

In general, the response to hindlimb unloading was limited to day 30. However, the response to HLU was modified in the Nox2(-/-) mice at both time points. This suggests that the effects of microgravity on immune populations may be regulated, in part, by Nox2. Furthermore, many of the changes noted (particularly the changes in percentages) were dominated by large KO-dependent increases in granulocyte counts. Low-dose radiation had very little impact on leukocyte populations at either time point.

Bibliography Type: Description: (Last Updated: 09/27/2021) 

Show Cumulative Bibliography Listing
 
Abstracts for Journals and Proceedings Mao XW. "Spaceflight and radiation induce microvessel and structure damage in ocular tissue." 9th Global Ophthalmology Summit, London, United Kingdom, March 15-16, 2017.

9th Global Ophthalmology Summit, London, United Kingdom, March 15-16, 2017. , Mar-2017

Abstracts for Journals and Proceedings Mao XW, Jones T, Campbell-Beachler M, Stanbouly S, Nishiyama N, Rodriguez D, Ortloff L, Mohanseenivasan V, Boerma M, Hauer-Jensen M, Nelson GA. "Center for Research on Cardiac and Vascular Effects of Space Radiation (Acute Risk Studies)." 2017 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 23-26, 2017.

2017 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 23-26, 2017. , Jan-2017

Articles in Other Journals or Periodicals Mao XW, Nishiyama N, Campbell-Beachler M, Gifford P, Haynes KE, Gridley DS, Pecaut M. "Role of NADPH oxidase as a mediator of oxidative damage in low-dose radiated and hindlimb-unloaded mice." Radiation Research. 2017 Aug 1. [Epub ahead of print] https://doi.org/10.1667/RR14754.1 ; PubMed PMID: 28763287 , Aug-2017
Articles in Peer-reviewed Journals Seawright JW, Samman Y, Sridharan V, Mao XW, Cao M, Singh P, Melnyk S, Koturbash I, Nelson GA, Hauer-Jensen M, Boerma M. "Effects of low-dose rate gamma-irradiation combined with simulated microgravity on markers of oxidative stress, DNA methylation potential, and remodeling in the mouse heart." PLoS One. 2017 Jul 5;12(7):e0180594. eCollection 2017. https://doi.org/10.1371/journal.pone.0180594 ; PubMed PMID: 28678877; PubMed Central PMCID: PMC5498037 , Jul-2017
Project Title:  Role of Oxidative Stress in Mediating the Effects of Combined Exposure to Simulated Microgravity and Radiation on Neurovascular Remodeling in Mouse Reduce
Fiscal Year: FY 2017 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Animal Biology: Vertebrate  
Start Date: 10/01/2013  
End Date: 08/31/2018  
Task Last Updated: 07/18/2016 
Download report in PDF pdf
Principal Investigator/Affiliation:   Mao, Xiao Wen  M.D. / Loma Linda University 
Address:  Radiation Medicine 
11175 Campus Street, Rm A1010, Chan Shun Pavilion 
Loma Linda , CA 92350-0001 
Email: xmao@llu.edu 
Phone: 909-558-8373  
Congressional District: 31 
Web:  
Organization Type: UNIVERSITY 
Organization Name: Loma Linda University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Gridley, Daila  Ph.D. Loma Linda University 
Hartman, Richard  Ph.D. Loma Linda University 
Pecaut, Michael  Ph.D. Loma Linda University 
Key Personnel Changes / Previous PI: July 2016 report: No changes.
Project Information: Grant/Contract No. NNX13AL97G 
Responsible Center: NASA ARC 
Grant Monitor: Sato, Kevin  
Center Contact: 650-604-1104 
kevin.y.sato@nasa.gov 
Solicitation / Funding Source: 2012 Space Biology NNH12ZTT001N 
Grant/Contract No.: NNX13AL97G 
Project Type: GROUND 
Flight Program:  
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Space Biology Element: (1) Animal Biology: Vertebrate
Space Biology Cross-Element Discipline: (1) Neurobiology
Space Biology Special Category: (1) Translational (Countermeasure) Potential
Flight Assignment/Project Notes: NOTE: End date changed to 8/31/2018 per F. Hernandez/ARC (Ed., 6/27/17)

NOTE: End date changed to 9/30/2017 per NSSC (Ed., 7/4/16)

Task Description: One of the main concerns for long-term deep manned space missions are health risks associated with altered gravitational environment and prolonged exposure to low-dose radiation above levels normally found on Earth. Microgravity and radiation exposure has been known to produce a number of neurological disturbances and neurodegeneration by space flight condition. However, the pathophysiological process from adaptive response to irreversible oxidative damage in the brain vasculature and the underlying mechanism(s) of these disturbances are less studied and remain unclear.

Our proposal seeks to fill in the gap by testing the hypothesis that NADPH oxidase is a critical source of the neurovascular oxidative stress following space flight conditions that mediates vascular remodeling in the brain, thus disrupting communication between endothelial cells and astrocytes and altering production of extracellular matrix (ECM) proteins. It is further proposed that these changes will contribute to increased vascular permeability and blood-brain barrier (BBB) disturbance, thus resulting in neurological deficit.

Our specific aims are 1) Define the causal relationships between space flight condition induced NADPH oxidase expression, vascular damage, and BBB function following microgravity and/or low-dose irradiation in mature mice using neuropathology, stereological, and automated image analysis, and neurobehavioral outcomes. 2) Determine if space flight condition-induced oxidative stress is mediated through NADPH oxidase in brain microvasculature.

Nox2 (a subunit of NADPH oxidase) gene knockout (Nox2(-/-)) mice, and wild-type (Nox2(+/+)) C57BL/6 mice will be used in this ground-based animal study. Hindlimb unloading (HLU) will be used to model the unloading, fluid shift, and physiological stress aspects of the microgravity component. Low-dose/low-dose-rate (LDR) gamma-irradiation (0.5 Gy at 0.01 cGy/h) will be delivered to the whole-body of mature adult mice to simulate the radiation component for over 21 days while the animals are tail-unloaded in cages for microgravity simulation. We will evaluate the radiation- and microgravity-induced brain vascular and tissue remodeling at multiple time points (1 day to 12 months post-irradiation).

Together, our unique, integrative, and quantitative activities with advanced imaging techniques, stereological analysis, and behavioral tests will provide insight into the molecular mechanisms of space flight condition-induced oxidative damage on brain tissue and vascular remodeling. Understanding how factors and environmental stress impact on vasculature, tissue remodeling, and function will increase our knowledge and focus toward more effective countermeasures during human space flight and planetary exploration. Our study will also lend new insights into the causes and possible treatments of debilitating neurovascular-related disease and neurodegeneration by targeting NADPH oxidase activation.

Research Impact/Earth Benefits: Oxidative stress in central nervous system (CNS) is a major contributor to brain injury and aging. There are strong indications that the physiological effects of space flight are similar to those seen in some neurodegenerative diseases and aging: multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease. Our study will provide the first detailed description of combined effects of microgravity and LDR radiation on oxidative stress-induced brain tissue and microvessel network remodeling and underlying mechanism(s) of potential interaction of space flight environmental components over a 12-month observation period. Our research will provide important input to elucidate cellular pathways of response and adaptation to stress imposed by environmental conditions in the brain vasculature. Understanding how factors and environmental insults impact on vasculature and tissue remodeling and function will increase our knowledge and help focus the approach toward more effective countermeasures during human space flight and planetary exploration. Our study might also lend new insights into the causes and possible treatments of debilitating neurovascular-related diseases and neurodegeneration.

Task Progress & Bibliography Information FY2017 
Task Progress: To determine whether NADPH oxidase-derived oxidative stress can account for unloading and radiation-induced deleterious effects on endothelial damage and neurovascular remodeling in a Nox2 knock-out (KO) mice model.

Wild-type (Nox2 (+/+)) C57BL/6 mice or Nox2 (-/-) (B6. 129S6-CYBBM) mice placed into one of the following groups (n=5-6/group): age-matched control, hindlimb unloading (HLU), low-dose/low-dose-rate radiation (LDR), or HLU+LDR simultaneously for 21 days, and were then sacrificed at day 7 or 1 month. Anti-orthostatic tail suspension was used to model the unloading, fluid shift, and physiological stress aspects of microgravity. The LDR was delivered using 57Co plates (0.04 Gy at 0.01 cGy/h) to the whole body in order to simulate the radiation experienced while in microgravity. Brains were isolated for characterization of various oxidative stress markers and vascular topology. Levels of 4-hydroxynonenal (4-HNE) protein, a specific marker for lipid peroxidation, were measured. Expression of aquaporin-4 (AQ4), a water channel protein expressed in astrocyte endfeet, was quantified. Long-term behavioral effects were also evaluated following chronic exposure of radiation + unloading. Thirty days after simulated space flight, KO mice showed decreased apoptosis (p<0.05) in the brain cortex compared to WT counterparts. The HLU-dependent increase in apoptosis in WT mice was not observed in KO mice. Level of 4-HNE protein was significantly elevated in the hippocampus after hindlimb unloading + radiation compared to controls in the WT mice (p<0.05), However, there was no significant differences between treated groups and control in the Nox2-KO mice at 7-day and 1-month time points. In contrast to findings in WT animals, superoxide dismutase level and expression of APQ4 was similar among all KO groups. For behavioral tests, KO mice that received irradiation spent significantly less time in the dark portion of the elevated zero maze than KO controls, suggesting abnormal exploratory/risk-taking behaviors (p<0.05). In summary, for most of the parameters, the oxidative response to HLU and LDR was suppressed in Nox2(-/-) mice at both time points. This suggests that NOX2-containing NADPH oxidase may contribute to space flight environment–induced oxidative stress.

To study the effect of simulated microgravity on metabolic change, brains were isolated at 1, 4, or 9 months after HLS exposure for analysis. Comparative untargeted metabolomics/ lipidomics profiling of the brain tissue was performed by ultra-performance liquid chromatography coupled with electro-spray quadrupole time of flight mass spectrometry (UPLC-ESI-QTOF-MS) to study specific changes that may be indicative of the adaptations in central nervous system (CNS) to space flight condition. Data was pre-processed using the XCMS software while the database search was performed using the Human Metabolome Database (HMDB), Madison Metabolomics Consortium Database (MMCD), LIPID MAPS and Metlin for putative metabolite identification. Although after a month following microgravity exposure, brains did not show significant differences in their metabolomes, we could detect them after longer periods of exposition. After 4 months, some metabolites like PI (18:3/0:0) and tetrahydroaldosterone-3-glucuronide went up (p<0.05) and some others such as PE (20:4/16:1), cardiolipin, PS (18:3/18:1), PE (20:3/14:0), PA (22:6/19:1), 11-peroxy-5Z,8Z,12E,14Z-eicosatetraenoate, and linoleamide were significantly (p<0.05) less abundant compared to the controls. After 9 months following microgravity exposure, some lipids including TG (14:1/18:4/18:4), TG (14:0/14:1/18:3), MG (18:0/0:0/0:0), PG (19:1/20:0), PE (22:4/P-18:0), PI (18:0/18:0), PS (18:3/22:0), and PS (22:4/22:1) went up (p<0.05) and some others, such as but-2-enoic acid, ganglioside GT2, LysoPE (20:3/0:0), galactoceramides and ceramides, diglycerides and phosphocholines, were significantly (p<0.05) downregulated in mice exposed to microgravity. In summary, our results show significant alterations in lipid profiles particularly glycerophospholipids class. For example, the levels of phospatidyl-ethanolamine and phosphatidyl-serine diminished significantly after four months following HLS in brain tissue which could be a result of increased lipid peroxidation, lipid degradation, or impaired lipid biogenesis in liver or export to the brain. These changes are likely to impact structural and chemical integrity of brain cells leading to structural and functional impairment. Characterization of longitudinal systemic changes in plasma of these mice is ongoing.

To compare changes in leukocyte parameters in wild-type (WT) and Nox2(-/-) knockout (KO) mice 30 and 120 days, Six-month-old female Nox2(+/+) and Nox2(-/-) C57BL/6 mice (n=4-6/group) were exposed to whole-body low-dose/low-dose-rate (LDR) gamma-irradiation using 57Co plates (0.04 Gy at 0.01 cGy/h) and/or hindlimb unloaded (HLU) for 21 days. Mice were sacrificed with 100% CO2 at 30 or 120 days after the simulated space flight period and blood was collected via cardiac puncture. An automated ABC Vet Hematology Analyzer was used to obtain white blood cell (WBC), lymphocyte (LYM), monocyte (MON), and granulocyte (GRA) counts and percentages.

Thirty days after simulated space flight, KO mice showed increased GRA counts (P<0.005) and decreased LYM (P<0.001) and MON (P<0.05) counts compared to WT controls. This resulted in a shift away from %LYM (P<0.001) and %MON (P<0.001) towards %GRA (P<0.001). Similarly, hindlimb unloading caused increases in WBC (P<0.05) and GRA counts (P<0.05), leading to proportional shifts away from %LYM (P=0.005) and %MON (P<0.05) towards %GRA (P<0.005). Interestingly, the HLU-dependent increase in GRA was augmented in the KO mice, resulting in a KO x HLU interaction (P<0.05). Furthermore, unloading increased %GRA and decreased %MON, but only in the KO mice, leading to significant KO x HLU interactions (Ps<0.05). Although %LYM were generally lower in KO mice, the LDR-dependent decrease in this parameter noted in WT was reversed in KO mice. This led to a significant KO x LDR interaction (P<0.05).

At 120 days after simulated-space flight, there were several significant main effects of KO due to increases in WBC (P<0.001), MON (P<0.001), and GRA (P<0.001) counts. Because the KO-dependent increases in GRA counts was much higher than in other populations, this led to a shift away from %LYM (P<0.001) and %MON (P<0.001) towards %GRA (P<0.001). In contrast to the response at 30 days, the only significant main effect of HLU at 120 days was a decrease in %GRA (P<0.05). However, the HLU-dependent increase on MON counts noted in WT mice was amplified in KO mice. The HLU-dependent decrease in LYM counts was reversed in KO mice. This led to significant KO x HLU interactions (Ps<0.05) in both of these parameters. Finally, the LDR-dependent increase in %MON noted in WT mice was not observed in KO mice, leading to a significant KO x LDR interaction (P<0.05).

In conclusion, low-dose radiation had very little impact on leukocyte populations at either time point. Furthermore, many of the changes noted (particularly the changes in percentages) were dominated by large KO-dependent increases in granulocyte counts. In general, the response to hindlimb unloading was limited to day 30. However, the response to HLU was modified in the Nox2(-/-) mice at both time points. This suggests that the effects of microgravity on immune populations may be regulated, in part, by Nox2.

Bibliography Type: Description: (Last Updated: 09/27/2021) 

Show Cumulative Bibliography Listing
 
Abstracts for Journals and Proceedings Cheema AK, Altadill T, Nelson G, Mao XW. "Simulated Microgravity Induces Changes in Brain Lipidome of Brain Tissue." Presented at 2016 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 8-11, 2016.

2016 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 8-11, 2016. , Feb-2016

Abstracts for Journals and Proceedings Mao XW, Nishiyama NC, Pecaut MJ, Campbell-Beachler M, Gifford P, Haynes K, Becronis C, Bellone J, Hartman R, Gridley DS. "Role of NADPH oxidase in neurovascular stress following unloading and/or low-dose radiation." 2016 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 8-11, 2016.

2016 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 8-11, 2016. , Feb-2016

Abstracts for Journals and Proceedings Nishiyama NC, Pecaut MJ, Gridley DS, Campbell-Beachler M, Gifford P, Mao XW. "Simulated Spaceflight Impacts Immune Populations in Nox2(-/-) Knockout Mice." International Society for Gravitational Physiology (ISGP), European Space Agency (ESA), Centre National d'Etudes Spatiales Joint Life Science Meeting ‘Life in Space for Life on Earth,’ Toulouse, France, June 5-10, 2016.

International Society for Gravitational Physiology (ISGP), European Space Agency (ESA), Centre National d'Etudes Spatiales Joint Life Science Meeting ‘Life in Space for Life on Earth,’ Toulouse, France, June 5-10, 2016. , Jun-2016

Abstracts for Journals and Proceedings Zheng J, Hauer-Jensen M, Boerma M, Nelson G, Mao XW. "Acute and late impact of simulated microgravity and low-dose radiation in the brain: a gene network analysis." 2016 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 8-11, 2016.

2016 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 8-11, 2016. , Feb-2016

Articles in Peer-reviewed Journals Mao XW, Nishiyama NC, Pecaut MJ, Campbell-Beachler M, Gifford P, Haynes KE, Becronis C, Gridley DS. "Simulated microgravity and low-dose/low-dose-rate radiation induces oxidative damage in the mouse brain." Radiat Res. 2016 Jun;185(6):647-57. http://dx.doi.org/10.1667/RR14267.1 ; PubMed PMID: 27243749 , Jun-2016
Articles in Peer-reviewed Journals Bellone JA, Gifford PS, Nishiyama NC, Hartman RE, Mao XW. "Long-term effects of simulated microgravity and/or chronic exposure to low-dose gamma radiation on behavior and blood–brain barrier integrity." npj Microgravity. 2016 Jun 9;2:16019. http://dx.doi.org/10.1038/npjmgrav.2016.19 , Jun-2016
Project Title:  Role of Oxidative Stress in Mediating the Effects of Combined Exposure to Simulated Microgravity and Radiation on Neurovascular Remodeling in Mouse Reduce
Fiscal Year: FY 2016 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Animal Biology: Vertebrate  
Start Date: 10/01/2013  
End Date: 09/30/2017  
Task Last Updated: 07/17/2015 
Download report in PDF pdf
Principal Investigator/Affiliation:   Mao, Xiao Wen  M.D. / Loma Linda University 
Address:  Radiation Medicine 
11175 Campus Street, Rm A1010, Chan Shun Pavilion 
Loma Linda , CA 92350-0001 
Email: xmao@llu.edu 
Phone: 909-558-8373  
Congressional District: 31 
Web:  
Organization Type: UNIVERSITY 
Organization Name: Loma Linda University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Gridley, Daila  Ph.D. Loma Linda University 
Hartman, Richard  Ph.D. Loma Linda University 
Pecaut, Michael  Ph.D. Loma Linda University 
Key Personnel Changes / Previous PI: July 2014 report: No changes.
Project Information: Grant/Contract No. NNX13AL97G 
Responsible Center: NASA ARC 
Grant Monitor: Taylor, Elizabeth  
Center Contact: 650.604.1783 
elizabeth.taylor-23@nasa.gov 
Solicitation / Funding Source: 2012 Space Biology NNH12ZTT001N 
Grant/Contract No.: NNX13AL97G 
Project Type: GROUND 
Flight Program:  
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Space Biology Element: (1) Animal Biology: Vertebrate
Space Biology Cross-Element Discipline: (1) Neurobiology
Space Biology Special Category: (1) Translational (Countermeasure) Potential
Flight Assignment/Project Notes: NOTE: End date changed to 9/30/2017 per NSSC (Ed., 7/4/16)

Task Description: One of the main concerns for long-term deep manned space missions are health risk associated with altered gravitational environment and prolonged exposure to low-dose radiation above levels normally found on Earth. Microgravity and radiation exposure has been known to produce a number of neurological disturbances and neurodegeneration by space flight condition. However, the pathophysiological process from adaptive response to irreversible oxidative damage in the brain vasculature and the underlying mechanism(s) of these disturbances are less studied and remain unclear.

Our proposal seeks to fill in the gap by testing the hypothesis that NADPH oxidase is a critical source of the neurovascular oxidative stress following space flight conditions that mediates vascular remodeling in the brain, thus disrupting communication between endothelial cells and astrocytes and altering production of extracellular matrix (ECM) proteins. It is further proposed that these changes will contribute to increased vascular permeability and blood-brain barrier (BBB) disturbance, thus resulting in neurological deficit.

Our specific aims are 1) Define the causal relationships between space flight condition induced NADPH oxidase expression, vascular damage, and BBB function following microgravity and/or low-dose irradiation in mature mice using neuropathology, stereological, and automated image analysis, and neurobehavioral outcomes. 2) Determine if space flight condition-induced oxidative stress is mediated through NADPH oxidase in brain microvasculature.

Nox2, (a subunit of NADPH oxidase) gene knockout (Nox2(-/-)) mice, and wild-type (Nox2(+/+)) C57BL/6 mice will be used in this ground-based animal study. Hindlimb unloading (HLU) will be used to model the unloading, fluid shift, and physiological stress aspects of the microgravity component. Low-dose/low-dose-rate (LDR) gamma-irradiation (0.5Gy at 0.01cGy/h) will be delivered to the whole-body of mature adult mice to simulate the radiation component for over 21 days while the animals are tailed-suspended in cages for microgravity simulation. We will evaluate the radiation- and microgravity-induced brain vascular and tissue remodeling at multiple time points (1 day to 12 months post-irradiation).

Together, our unique, integrative, and quantitative activities with advanced imaging techniques, stereological analysis, and behavioral tests will provide insight into the molecular mechanisms of space flight condition-induced oxidative damage on brain tissue and vascular remodeling. Understanding how factors and environmental stress impact on vasculature, tissue remodeling, and function will increase our knowledge and focus toward more effective countermeasures during human space flight and planetary exploration. Our study will also lend new insights into the causes and possible treatments of debilitating neurovascular-related disease and neurodegeneration by targeting NADPH oxidase activation.

Research Impact/Earth Benefits: Oxidative stress in central nervous system (CNS) is a major contributor to brain injury and aging. There are strong indications that the physiological effects of space flight are similar to those seen in some neurodegenerative diseases and aging: multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease. Our study will provide the first detailed description of combined effects of microgravity and LDR radiation on oxidative stress-induced brain tissue and microvessel network remodeling and underlying mechanism(s) of potential interaction of space flight environmental components over a 12-month observation period. Our research will provide important input to elucidate cellular pathways of response and adaptation to stress imposed by environmental conditions in the brain vasculature. Understanding how factors and environmental insults impact on vasculature and tissue remodeling and function will increase our knowledge and help focus the approach toward more effective countermeasures during human space flight and planetary exploration. Our study might also lend new insights into the causes and possible treatments of debilitating neurovascular-related diseases and neurodegeneration.

Task Progress & Bibliography Information FY2016 
Task Progress: Experiments and data analysis are ongoing as planned. So far, we have finished four batches of animal studies for specific aim 1, at 7 days, 1, 3, and 9 month time points. We also started Nox2 knockout mice study as proposed in specific aim 2. Both 7 day and 1 month time points study are ongoing. We examined the gene expression using microarray analysis, protein markers associated with the induction of oxidative stress and apoptosis in the brain, metabolic response using metabolic biochemical assays, hematological parameters and behavioral responses after combined exposure to simulated microgravity and low-dose radiation at day 7 and 9 months.

To study the role of oxidative stress in the mouse brain after simulated microgravity and low-dose radiation exposure, the level of 4-HNE protein was significantly elevated in the hippocampus after hindlimb unloading + radiation compared to controls (p<0.05). Unloading + radiation group had highest level of NOX2 expression compared to controls (p<0.05). There was a significant increase in SOD protein expression in the group that received unloading or combination of unloading + radiation compared to control (P<0.05). SOD expression also showed a strong trend for an interaction between unloading and radiation (p=0.09). Significantly higher levels of AQ4 production in the mouse hippocampus were also noted after combined exposure or unloading only compared to controls (p<0.05). This study suggests that hindlimb unloading and radiation may induce oxidative stress in mouse brain. Nox2-containing nicotinamide adenosine dinucleotide phosphate (NADPH oxidase) may contribute to combined exposure to simulated microgravity and low-dose radiation–induced oxidative stress. The long-term impact of combined exposure to simulated microgravity and low-dose radiation-induced oxidative stress will be examined in additional time points in future studies.

A gene network analysis was performed to provide information on biological pathways underlying the impact of combined exposure. Total RNA samples were extracted from brains and processed for gene expression microarray. The activation/inhibition (¦Z-score¦=2, p-value =0.05) of Global Canonical Pathways was analyzed by Ingenuity Pathway Analysis (IPA). At day 7, there was no significant activation/inhibition of any pathways after HLU, LD gamma R or the combination of both. At 9 months, HLU induced significant activations of HMGB1 signaling, a risk factor for memory impairment and chronic neurodegeneration. Cellular immune response pathways including PKC theta signaling, PI3K signaling, as well as the production of nitric oxide and reactive oxygen species (ROS) signaling were also activated. PPAR signaling was significantly inhibited at 9 months after HLU. The combination of HLU and LD gamma R induced significant activations of HMGB1 and Neuregulin signaling only. The mRNA expression of many genes that regulated oxidative stress (e.g., MPO, SERPINA1, NOXa1), extracellular matrix remodeling (e.g., ADAM8, COL9a), endothelial cell biology (e.g., ITGA5, MMP1), and inflammation (e.g., SOS1, TNFRSF1A) were significantly altered (p<0.05) after HLU or HLU+ LD gamma R compared to control. Our findings provide candidate genes and biological pathways underlying phenotypes induced by HLU and LD/LDR. These data indicate that space environmental factors may have an impact on pathological and functional consequences associated with late neurodegeneration.

To study the impact of long-term behavioral effects of chronic exposure of low-dose radiation and simulated microgravity, Mice then underwent a series of behavioral tests at 4 time-points (1 week, 1 month, 4 months, and 8 months post-exposure) to assess anxiety-related behaviors (elevated zero maze), sensorimotor coordination and balance (rotarod), exploratory behavior/activity levels (open field), learned helplessness/depression-like behavior (tail suspension test), and spatial learning/memory (water maze). Mice that received hindlimb unloading (i.e., the unloading and combination groups) were hypoactive in the open field compared to the radiation-only group. Mice that received a combination of unloading and irradiation spent significantly less time in the dark portion of the elevated zero maze than controls, suggesting abnormal exploratory/risk-taking behaviors. No differences were observed on the water maze, rotarod, or tail suspension tests. These results suggest that microgravity and/or a chronic, low dose of gamma radiation may lead to changes in exploratory/risk-taking behaviors, but that neither cause deficits in spatial learning, coordination and balance, nor learned helplessness/depression-like behavior. Such findings have implications for extended manned space missions planned in the near future.

We also examined time-dependent changes of hematological parameters after hindlimb unloading and low-Dose/low-dose-rate radiation. Blood was collected via cardiac puncture. An automated ABC Hematology Analyzer (scil Vet, Inc.) was used to obtain white blood cell (WBC), red blood cell (RBC), and platelet (PLT) measurements. There were no significant main effects of day, radiation, or unloading on the count of any major immune subset. However, significant day-dependent decreases were observed in lymphocyte (P<0.001) and monocyte (P<0.001) percentages, with corresponding increases in granulocytes (P<0.001), suggesting an effect of age. In erythrocytes, there were decreases noted in the combined treatment group (LDR+HLU) on day 7 post-exposure, and increases in all non-control groups on day 30 for RBC, hemoglobin (HGB) levels, and hematocrit (HCT). This led to main effects of day in RBC, HGB, and HCT (P<0.001) and a Day x HLU interaction on HGB (P<0.05). Furthermore, with the exception of day 30, the LDR+HLU group was consistently low in all three parameters at other time points, resulting in significant LDR x HLU interactions (P<0.05) in HGB and HCT. Similarly, RBC distribution width (RDW) is increased at the early time points in the unloading groups, resulting in significant main effects of day (P<0.001) and HLU (P<0.001), as well as a Day x HLU interaction (P=0.001). There were no significant effects or interactions involving day, radiation, or unloading for platelet count or volume. The long term impact of the spaceflight environment on immune populations was minimal. The early decreases in the LDR+HLU group noted in RBC, HGB, and HCT, along with increases in RDW, are consistent with reports that spaceflight leads to anemia. Furthermore, the increases noted in erythrocyte parameters on day 30 suggest that both microgravity and radiation may cause a change in hematopoiesis, resulting in overcompensation. Although the effects of simulated spaceflight appear to be mild on these measures, further study is required to determine the functional consequences of these changes.

Bibliography Type: Description: (Last Updated: 09/27/2021) 

Show Cumulative Bibliography Listing
 
Abstracts for Journals and Proceedings Burkhart KA, Brooks DJ, Bouxsein ML, Mao XW. "Combined effect of hindlimb unloading and radiation on trabecular and cortical bone in adult mice." Presented at the 2015 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 13-15, 2015.

2015 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 13-15, 2015. , Jan-2015

Abstracts for Journals and Proceedings Mao XW, Pecaut MJ, Campbell-Beachler M, Gifford P, Nishiyama N, Gridley DS. "Role of Oxidative Stress in Combined Effects of Hindlimb Unloading and Low-dose Radiation in Mouse Brain." 15th International Congress of Radiation Research (ICRR), Kyoto, Japan. May 25-29, 2015.

15th International Congress of Radiation Research, Kyoto, Japan. May 25-29, 2015. , May-2015

Abstracts for Journals and Proceedings Mao YX, Nishiyama N, Pecaut MJ, Campbell-Beachler M, Gifford P, Gridley DS. "Simulated microgravity and low-dose radiation induces oxidative damage in the mouse hippocampus." American Society for Gravitational and Space Research (ASGSR), Alexandria, VA November 11-14 2015.

31st Annual Meeting of the American Society for Gravitational and Space Research, Alexandria, VA, November 11-14, 2015. , Nov-2015

Project Title:  Role of Oxidative Stress in Mediating the Effects of Combined Exposure to Simulated Microgravity and Radiation on Neurovascular Remodeling in Mouse Reduce
Fiscal Year: FY 2015 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Animal Biology: Vertebrate  
Start Date: 10/01/2013  
End Date: 09/30/2016  
Task Last Updated: 07/25/2014 
Download report in PDF pdf
Principal Investigator/Affiliation:   Mao, Xiao Wen  M.D. / Loma Linda University 
Address:  Radiation Medicine 
11175 Campus Street, Rm A1010, Chan Shun Pavilion 
Loma Linda , CA 92350-0001 
Email: xmao@llu.edu 
Phone: 909-558-8373  
Congressional District: 31 
Web:  
Organization Type: UNIVERSITY 
Organization Name: Loma Linda University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Gridley, Daila  Ph.D. Loma Linda University 
Hartman, Richard  Ph.D. Loma Linda University 
Pecaut, Michael  Ph.D. Loma Linda University 
Key Personnel Changes / Previous PI: July 2014 report: No changes.
Project Information: Grant/Contract No. NNX13AL97G 
Responsible Center: NASA ARC 
Grant Monitor: Smith, Jeffrey  
Center Contact: 650-604-0880 
jeffrey.d.smith2@nasa.gov 
Solicitation / Funding Source: 2012 Space Biology NNH12ZTT001N 
Grant/Contract No.: NNX13AL97G 
Project Type: GROUND 
Flight Program:  
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Space Biology Element: (1) Animal Biology: Vertebrate
Space Biology Cross-Element Discipline: (1) Neurobiology
Space Biology Special Category: (1) Translational (Countermeasure) Potential
Task Description: One of the main concerns for long-term deep manned space missions are health risk associated with altered gravitational environment and prolonged exposure to low-dose radiation above levels normally found on Earth. Microgravity and radiation exposure has been known to produce a number of neurological disturbances and neurodegeneration by space flight condition. However, the pathophysiological process from adaptive response to irreversible oxidative damage in the brain vasculature and the underlying mechanism(s) of these disturbances are less studied and remain unclear.

Our proposal seeks to fill in the gap by testing the hypothesis that NADPH oxidase is a critical source of the neurovascular oxidative stress following space flight conditions that mediates vascular remodeling in the brain, thus disrupting communication between endothelial cells and astrocytes and altering production of extracellular matrix (ECM) proteins. It is further proposed that these changes will contribute to increased vascular permeability and blood-brain barrier (BBB) disturbance, thus resulting in neurological deficit.

Our specific aims are 1) Define the causal relationships between space flight condition induced NADPH oxidase expression, vascular damage, and BBB function following microgravity and/or low-dose irradiation in mature mice using neuropathology, stereological, and automated image analysis, and neurobehavioral outcomes. 2) Determine if space flight condition-induced oxidative stress is mediated through NADPH oxidase in brain microvasculature.

Nox2, (a subunit of NADPH oxidase) gene knockout (Nox2(-/-)) mice, and wild-type (Nox2(+/+)) C57BL/6 mice will be used in this ground-based animal study. Hindlimb suspension will be used to model the unloading, fluid shift, and physiological stress aspects of the microgravity component. Low-dose/low-dose-rate (LDR) gamma-irradiation (0.5Gy at 0.01cGy/h) will be delivered to the whole-body of mature adult mice to simulate the radiation component for over 21 days while the animals are tailed-suspended in cages for microgravity simulation. We will evaluate the radiation- and microgravity-induced brain vascular and tissue remodeling at multiple time points (1day to 12 months post-irradiation).

Together, our unique, integrative, and quantitative activities with advanced imaging techniques, stereological analysis, and behavioral tests will provide insight into the molecular mechanisms of space flight condition-induced oxidative damage on brain tissue and vascular remodeling. Understanding how factors and environmental stress impact on vasculature, tissue remodeling, and function will increase our knowledge and focus toward more effective countermeasures during human space flight and planetary exploration. Our study will also lend new insights into the causes and possible treatments of debilitating neurovascular-related disease and neurodegeneration by targeting NADPH oxidase activation.

Research Impact/Earth Benefits: Oxidative stress in central nervous system (CNS) is a major contributor to brain injury and aging. There are strong indications that the physiological effects of space flight are similar to those seen in some neurodegenerative diseases and aging: multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease. Our study will provide the first detailed description of combined effects of microgravity and LDR radiation on oxidative stress-induced brain tissue and microvessel network remodeling and underlying mechanism(s) of potential interaction of space flight environmental components over a 12-month observation period. Our research will provide important input to elucidate cellular pathways of response and adaptation to stress imposed by environmental conditions in the brain vasculature. Understanding how factors and environmental insults impact on vasculature and tissue remodeling and function will increase our knowledge and help focus the approach toward more effective countermeasures during human space flight and planetary exploration. Our study might also lend new insights into the causes and possible treatments of debilitating neurovascular-related diseases and neurodegeneration.

Task Progress & Bibliography Information FY2015 
Task Progress: Three batches of studies have been performed for 7 days, 1 and 12 months time points. LDR gamma-irradiation using 57Co plates (0.04Gy at 0.01cGy/h) was delivered to the whole-body of mature 6 month old adult C57BL/6 mice (n=4-6/group) to simulate the radiation component. Anti-orthostatic tail suspension was used to model the unloading, fluid shift, and physiological stress aspects of the microgravity component. Mice were hindlimb suspended and/or irradiated for 21 days. To examine the induction of apoptosis-associated protein profiles in the brain after combined exposure to simulated microgravity and low-dose radiation, brain tissues were isolated for characterization of apoptosis-associated proteins 7 days after the completion of irradiation and unloading. Brain cell lysates from each group were incubated overnight with RayBio Human Apoptosis Antibody Array Membranes. Biotinylated antibodies were used to detect bound proteins and signals were visualized by chemiluminescence. The results were subjected to two-way analysis of variance (ANOVA). BAD, FAS, and FASL proteins were significantly activated (p<0.05) at the 7-day time point in the unloaded group compared to controls. There were statistical trends for differences between groups noted in BAX, BCL-2, and HSP27 (P<0.1), where hindlimb unloaded mice had a greater than 2-3 fold higher expression level compared with radiation alone or unloaded + radiation groups. These proteins play important roles in induction of apoptosis. There was also a trend for increase in the level of anti-apoptotic protein CD40L in the unloaded + radiation group compared to other groups. This study demonstrates that hindlimb unloading may induce early apoptosis in mouse brain and radiation may exacerbate simulated microgravity-induced damage in brain structure and tissue function. Combined exposure to simulated microgravity and low-dose radiation-induced apoptosis will be examined for longer time points in our future studies.

To study the impact of combined exposure on hematological parameters, blood was collected in K2-EDTA coated syringes via cardiac puncture and analysed via the scil Vet ABC Hematology Analyzer. Although there was no significant main effects of either parameter on the count of any major immune subset, there were trends for suspension x interactions for WBC, lymphocyte, and granulocyte counts (P<0.1). In all cases, this was due to decreases noted in the suspension alone or radiation alone groups that was not present in the combined treatment group. While there were no effects of LD/LDR radiation on the proportions of each population, there was a suspension-induced proportional shift away from lymphocytes (P<0.05) toward granulocytes (P<0.1). While there were overall suspension-induced decreases in erythrocyte count and hemoglobin levels (P<0.05), these decrease primarily occurred in mice that also received LD/LDR radiation, resulting in significant suspension x radiation interactions (P<0.05). A similar interaction was noted in hematocrit (P<0.05). There were also suspension-induced increases in both mean corpuscular volume and RBC distribution width (P<0.05), with a decreases in mean corpuscular hemoglobin concentration (P<0.1). There was only a trend for a radiation-induced increase in mean corpuscular hemoglobin (P<0.1). There was no impact of either stressor on platelet count or volume.

To study the behavioral response to combined exposure of simulated microgravity and radiation, the mice went through a series of behavioral tests to assess anxiety-related behaviors (elevated zero maze), sensorimotor coordination and balance (rotarod), exploratory behavior and activity levels (open field), and spatial learning / memory (water maze). Preliminary findings show that the all groups performed similarly in the elevated zero maze. Mice exposed to radiation only fell off the rotarod more quickly (p < 0.05) compared with control, unloading only, or unloading+irradiation mice (which did not differ). However, all treatment groups trended toward less activity than controls during open field testing and demonstrated spatial learning and/or memory deficits in the water maze. Thus, 3 weeks of Co-57 irradiation, hindlimb unloading, or a combination of both led to sensorimotor coordination and balance deficits, hypoactivity, and spatial learning / memory deficits. Overall, our study demonstrates that hindlimb unloading may induce early changes in mouse brain structure and function and radiation may exacerbate simulated microgravity-induced damage. Our data indicate that environmental factors have differentials responses in immunocytes, erythrocytes, and platelets. These results suggest that multiple factors present in the long-term deep-space environment may have detrimental effects on an astronaut’s motor and cognitive abilities.

Tissues from this study have been shared with collaborators at various groups and institutes for evaluation and analysis.

Bibliography Type: Description: (Last Updated: 09/27/2021) 

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Abstracts for Journals and Proceedings Mao YX, Pecaut MJ, Campbell-Beachler M, Gifford P, Gridley DS. "Detection of Early-stage Apoptosis in Mouse Brain after Combined Exposure to Simulated Microgravity and Radiation." 30th Annual Meeting of the American Society for Gravitational and Space Research, Pasadena, CA, October 22-26, 2014.

Program and abstracts. 30th Annual Meeting of the American Society for Gravitational and Space Research, Pasadena, CA, October 22-26, 2014. In press as of September 2014. , Sep-2014

Abstracts for Journals and Proceedings Sanders K, Bellone JA, Montanari R, Gifford P, Hartman RE, Mao XW. "Behavioral response to combined exposure of simulated microgravity and radiation." Neuroscience 2014, Washington, DC, November 15-19, 2014.

Neuroscience 2014, Washington, DC, November 15-19, 2014. Program#/Poster#: 360.07/SS66. Available at http://www.abstractsonline.com/Plan/ViewAbstract.aspx?mID=3527&sKey=ae2f7f6d-6371-4fdd-aa1f-4348a1849a1a&cKey=afca8d63-5ed1-420b-869e-588f4e26a99e&mKey=54c85d94-6d69-4b09-afaa-502c0e680ca7 ; accessed 11/25/14. , Nov-2014

Abstracts for Journals and Proceedings Pecaut MJ, Gridley DS, Campbell-Beachler M, Gifford P, Mao XW. "Impact of combined exposure to anti-orthostatic tail suspension and low-dose/low-dose-rate radiation on hematological parameters." 30th Annual Meeting of the American Society for Gravitational and Space Research, Pasadena, CA, October 22-26, 2014.

Program and abstracts. 30th Annual Meeting of the American Society for Gravitational and Space Research, Pasadena, CA, October 22-26, 2014. In press as of September 2014. , Sep-2014

Project Title:  Role of Oxidative Stress in Mediating the Effects of Combined Exposure to Simulated Microgravity and Radiation on Neurovascular Remodeling in Mouse Reduce
Fiscal Year: FY 2014 
Division: Space Biology 
Research Discipline/Element:
Space Biology: Animal Biology: Vertebrate  
Start Date: 10/01/2013  
End Date: 09/30/2016  
Task Last Updated: 07/26/2013 
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Principal Investigator/Affiliation:   Mao, Xiao Wen  M.D. / Loma Linda University 
Address:  Radiation Medicine 
11175 Campus Street, Rm A1010, Chan Shun Pavilion 
Loma Linda , CA 92350-0001 
Email: xmao@llu.edu 
Phone: 909-558-8373  
Congressional District: 31 
Web:  
Organization Type: UNIVERSITY 
Organization Name: Loma Linda University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Gridley, Daila  Ph.D. Loma Linda University 
Hartman, Richard  Ph.D. Loma Linda University 
Pecaut, Michael  Ph.D. Loma Linda University 
Project Information: Grant/Contract No. NNX13AL97G 
Responsible Center: NASA ARC 
Grant Monitor: Smith, Jeffrey  
Center Contact: 650-604-0880 
jeffrey.d.smith2@nasa.gov 
Solicitation / Funding Source: 2012 Space Biology NNH12ZTT001N 
Grant/Contract No.: NNX13AL97G 
Project Type: GROUND 
Flight Program:  
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Space Biology Element: (1) Animal Biology: Vertebrate
Space Biology Cross-Element Discipline: (1) Neurobiology
Space Biology Special Category: (1) Translational (Countermeasure) Potential
Task Description: One of the main concerns for long-term deep manned space missions are health risk associated with altered gravitational environment and prolonged exposure to low-dose radiation above levels normally found on Earth. Microgravity and radiation exposure has known to produce a number of neurological disturbances and neurodegeneration by space flight condition. However, the pathophysiological process from adaptive response to irreversible oxidative damage in the brain vasculature and the underlying mechanism(s) of these disturbances are less studied and remain unclear.

Our proposal seeks to fill in gap by testing the hypothesis that NADPH oxidase is a critical source of the neurovascular oxidative stress following space flight condition that mediates vascular remodeling in the brain, thus disrupting communication between endothelial cells and astrocytes and altering production of extracellular matrix (ECM) proteins. It is further proposed that these changes will contribute to increased vascular permeability and blood-brain barrier (BBB) disturbance, thus resulting in neurological deficit.

Our specific aims are 1) Define the causal relationships between space flight condition induced NADPH oxidase expression, vascular damage, and BBB function following microgravity and/or low-dose irradiation in mature mice using neuropathology, stereological, and automated image analysis, and neurobehavioral outcomes. 2) Determine if space flight condition-induced oxidative stress is mediated through NADPH oxidase in brain microvasculature.

Nox2, (a subunit of NADPH oxidase) gene knockout (Nox2(-/-)) mice and wild-type (Nox2(+/+)) C57BL/6 mice will be used in this ground-based animal study. Hindlimb suspension will be used to model the unloading, fluid shift, and physiological stress aspects of the microgravity component. Low-dose/low-dose-rate (LDR) gamma-irradiation (0.5Gy at 0.01cGy/h) will be delivered to the whole-body of mature adult mice to simulate the radiation component for over 21 days while the animals are tailed-suspended in cages for microgravity simulation. We will evaluate the radiation- and microgravity-induced brain vascular and tissue remodeling at multiple time points (1day to 12 months post-irradiation).

Together, our unique, integrative, and quantitative activities with advanced imaging techniques, stereological analysis, and behavioral tests will provide insight into the molecular mechanisms of space flight condition-induced oxidative damage on brain tissue and vascular remodeling. Understanding how factors and environmental stress impact on vasculature, tissue remodeling, and function will increase our knowledge and focus toward more effective countermeasures during human space flight and planetary exploration. Our study will also lend new insights into the causes and possible treatments of debilitating neurovascular-related disease and neurodegeneration by targeting NADPH oxidase activation.

Research Impact/Earth Benefits: 0

Task Progress & Bibliography Information FY2014 
Task Progress: New project for FY2014.

Bibliography Type: Description: (Last Updated: 09/27/2021) 

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 None in FY 2014