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Mol Brain
Mol Brain
Molecular Brain
1756-6606
BioMed Central London

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10.1186/s13041-024-01133-2
Micro Report
Simulated weightlessness procedure, head-down bed rest has reversible effects on the metabolism of rhesus macaque
Li Yuting 1
http://orcid.org/0000-0002-0126-5671
Zhang Xu zhangxubiao@sjtu.edu.cn

1
Xu Zhen 1
Chu Xixia 1
Hu Zhiqiang 1
Ye Zhengyang 1
Li Caiqin 1
Wang Zhenbo 1
Zeng Bin 2
Pan Jingyu 2
Zhao Qian 1
Zhou Chengbin 3
Lan Zhaohui 1
Kan Guanghan 2
He Guang 1
Xu Xiaodan xuxiaodan.xuan@163.com

2
http://orcid.org/0000-0001-5384-4838
Li Weidong liwd@sjtu.edu.cn

14
1 grid.16821.3c 0000 0004 0368 8293 Bio-X Institutes (Key Laboratory for the Genetics of Development and Neuropsychiatric Disorders, Brain Health and Brain Technology Research Center in Global Institute of Future Technology, Institute of Psychology and Behavioral Science, Ministry of Education), Shanghai Jiao Tong University, Shanghai, 200240 China
2 https://ror.org/001ycj259 grid.418516.f 0000 0004 1791 7464 National Key Laboratory of Human Factors Engineering, Astronaut Research and Training Center, Beijing, 100094 China
3 grid.16821.3c 0000 0004 0368 8293 Department of Neurosurgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025 China
4 WLA Laboratories, World Laureates Association, Shanghai, 201203 China
3 9 2024
3 9 2024
2024
17 6510 5 2024
7 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
It is a consensus in the international manned space field that factors such as microgravity during the space flight can cause anxiety, depression and other important brain function abnormalities in astronauts. However, the neural mechanism at the molecular level is still unclear. Due to the limitations of research conditions, studies of biological changes in the primate brain have been comparatively few. We took advantage of -6° head-down bed rest (HDBR), one of the most implemented space analogues on the ground, to investigate the effects of simulated weightlessness on non-human primate brain metabolites. The Rhesus Macaque monkeys in the experiment were divided into three groups: the control group, the 42-day simulated weightlessness group with HDBR, and the recovery group, which had 28 days of free activity in the home cage after the HDBR. Liquid chromatography-mass spectrometry (LC-MS) was used to perform metabolomics analysis on specific brain areas of the monkeys under three experimental conditions. Our results show that simulated weightlessness can cause neurotransmitter imbalances, the amino acid and energy metabolism disorders, and hormone disturbances. But these metabolomics changes are reversible after recovery. Our study suggests that long-term brain damage in space flight might be reversible at the metabolic level. This lays a technical foundation for ensuring brain health and enhancing the brain function in future space studies.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13041-024-01133-2.

Keywords

Simulated weightlessness
Rhesus macaque
Metabolomics
National Key Research and Development Program of China2018YFE0126700 Li Weidong Shanghai Education Commission Research and Innovation Program2019-01-07-00-02-E00037 Li Weidong Shanghai Municipal Commission of Science and Technology Program21dz2210100 2021SHZDZX Li Weidong the “111” Program of Higher Education Discipline Innovationnone Li Weidong http://dx.doi.org/10.13039/501100002858 China Postdoctoral Science Foundation 2021M702137 Zhang Xu Natural Science Foundation of Chongqingcstc2021jcyj-msxmX1176 2022NSCQ-MSX1304 Zhang Xu Lan Zhaohui issue-copyright-statement© Min Zhuo, Bong-Kiun Kaang and BioMed central Ltd. 2024
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pmcMain text

During long-term spaceflight, astronauts experience fluid redistribution due to microgravity, which can lead to problems in many body systems. The impact on the brain might be particularly severe when astronauts potentially experiencing cognitive impairments, such as spatial orientation and learning and memory difficulties. However, human understanding of these effects remains limited, and the neural mechanism at the molecular level is still unclear. The impact of microgravity on cognition including spatial orientation, motion perception, object recognition, learning, and memory. Damage to neuronal synapses and inhibition of neurogenesis in the hippocampus are considered to be the main mechanisms by which microgravity affects cognition [1]. Other studies have also shown that the dorsomedial prefrontal cortex (dmPFC) plays an important role in cognitive control and emotion regulation [2]. Considering all these findings, we determined that the hippocampus and dmPFC might be closely related to the mechanism of brain damage in microgravity. Metabolomics research plays an important role in revealing the changes of microgravity on the molecular level of the brain and explaining the impact of microgravity on brain cognition and other functions. The NASA Human Research Program indicates that metabolites associated with genetic toxicity stress, inflammation, and amino acid metabolism changes during long-term spaceflight [3]. Due to the limitations of research conditions, studies of biological changes in the primate brain have been comparatively few. Alternatively, the head-down bed rest (HDBR) study and non-human primates are widely used to simulate weightlessness on the ground [4]. Rhesus Macaque monkeys, which are closely related to humans and widely used in system neuroscience [5], were chosen for the HDBR experiment.

Therefore, in our experiment, we chose the hippocampus and dmPFC samples from Rhesus Macaque monkeys for mass spectrometry analysis to determine the metabolic status, which can be used as a preliminary assessment of brain damage. Fifteen Rhesus Macaque monkeys in the experiment were divided into three groups: the control group, the 42-day simulated weightlessness group with HDBR, and the recovery group, which had 28 days of free activity in the home cage after the HDBR (Fig. 1A, Figure S1A). The liquid chromatography-mass spectrometry (LC-MS), combined with high-throughput labeling detection, can determine the relative molecular mass of different metabolic products and conjecture chemical formula, and structural formula of a compound. Thousands of metabolic features can be simultaneously detected and quantified, based on mass spectrometry metabolomics methods (Figure S1B-E). Our results show that there is a great number of different metabolites between the HDBR group and the control group (Fig. 1B). The enriched pathways are mainly related to amino acid metabolism, energy metabolism, and hormone levels (Fig. 1C-D). Among them, the disruption of energy metabolism is closely related to cognitive impairments caused by exposure to microgravity, as impaired brain energy metabolism can affect an individual’s cognitive abilities [6]. There are also reports of morphological and metabolic changes in rat hippocampal neurons’ mitochondria after simulating 28 days of microgravity exposure [7].

Fig. 1 HDBR has reversible effects on the metabolism of rhesus macaque. A Schematic diagram. Schematic diagram of grouping and experimental procedure. B Volcano Plot. Log2 (fold change) is the abscissa, and the negative logarithm of q-value -log10 (q-value) is the ordinate. Points with p-value greater than 0.05 are gray; points with fold change between 0.8 and 1.2 and p-value less than 0.05 are blue; points with fold change less than 0.8 or greater than 1.2 and p-value less than 0.05 are red. C-D KEGG Pathway. Metabolic pathways show the top 20 rows of the metabolic pathways annotated for the differential metabolites. Ordinate is the metabolic pathway name. Count indicates the number of metabolites in this metabolic pathway. E Ordinate is the significantly different metabolites. F Heat map of the cluster analysis. Each row in the figure represents a differential ion, and each column represents a sample. Different colors indicate different intensities, and colors range blue to red, indicating strength from low to high. G Venn diagram. H Screening of differential ions between groups. The project used multivariate analysis of the VIP values of the first two principal components of the PLS-DA model, combined with univariate analysis of fold-change and q-value values to screen differentially expressed metabolites. Screening conditions: (1) fold change > 1.2 or < 0.8; (2) q-value < 0 0.05. Taking the intersection of these two criteria yields the shared ion, which represents the differential ion. I-J Volcano Plot. K KEGG Pathway

Interestingly, we found that several metabolites were significantly enriched in the D-amino acids synthesis and metabolism pathways in the simulated microgravity group compared to the control group (Fig. 1C-D). In a rodent tail suspension research, which is another microgravity simulating model, shows that amino acid metabolism disorder is an important mechanism for inducing depressive states under simulated microgravity [8]. The results of disrupted metabolism of glycine, tryptophan, arginine, proline, and phenylalanine in our research are consistent with the results of amino acid metabolism pathway alterations in rodent simulated microgravity condition [9]. In the mammalian brain, D-amino acids present in neuroendocrine and endocrine tissues and act as co-agonists of N-methyl-D-aspartate (NMDA) glutamate receptors, which is responsible for learning, memory, and behavior. D-amino acids are also one of the main regulators of adult neurogenesis [10]. This is consistent with our previous findings that simulated microgravity with a HDBR reduces neurogenesis in the brains of rhesus monkeys [1]. Our results indicate that the disruption of D-amino acid metabolism may be an important metabolic change resulting from simulated microgravity conditions that lead to cognitive changes.

In addition, the neuroactive ligand-receptor interaction pathway was also identified as an enriched pathway in the HDBR group, compare to the control group (Fig. 1C-D). This pathway mainly involves metabolites related to neurotransmitters (Fig. 1E), which are chemical substances that transmit information between neurons and play a crucial role in the brain. The heat map of the cluster analysis also shows significant differences in metabolites in the dmPFC and hippocampus regions before and after simulated weightlessness (Fig. 1F).

Among neuroactive ligand-receptor interaction pathway, neurotransmitters such as epinephrine, serotonin, and N-acetylaspartylglutamic acid showed significant differences, and these neurotransmitters are closely related to memory. Furthermore, our research results show that L-aspartic acid in the hippocampal brain region of the HDBR group was halved compared to the control group, and the difference was significant. L-aspartic acid is an important excitatory neurotransmitter and has been found decreased in patients with depression and brain atrophy [11]. This is like previous findings on cognitive and emotional effects in space, where recent astronaut experiments reported long-term negative effects on cognition and emotion [12].

At the hormone level, we found the expression of cortisol decreases in the mPFC region in HDBR Group (Fig. 1E). Cortisol is a stress-related hormone and is related to cognition [13]. We also found HDBR can lead to a significant increase in the expression levels of two important mammalian hormones, adrenaline and norepinephrine, in the hippocampus of macaques. This might be the result of the hypothalamic–pituitary–adrenal (HPA) axis responding to the stress in HDBR procedure [14].

Furthermore, we found that compared to the control group, Uridine 5’-diphosphate showed significant changes in the hippocampus and dmPFC regions of the HDBR group, which may indicate that simulated microgravity condition causes neuronal cell damage. Uridine 5’-diphosphate is closely related to neuron damage and plays an important role in the process of glial cells participating in the clearance of dead cells and their debris [15].

It is worth noticing that the differential metabolites in the neuroactive ligand-receptor interaction pathway returned to normal levels after the recovery period (Fig. 1G-J), and was no longer an enriched metabolites pathway (Fig. 1K). Therefore, we consider that the concentration changes in the neuroactive ligand-receptor interaction pathway in the hippocampus and dmPFC tissues provide a potential molecular-level explanation for the cognitive and decision-making abnormalities induced by simulated microgravity.

Furthermore, after the recovery period, there were fewer differential metabolites compared to the control group, mainly concentrated in pathways related to amino acid metabolism, energy metabolism, and hormone levels (Fig. 1K). These differential metabolites were not shared with the simulated weightlessness group, indicating that the 28-day recovery period after HDBR might be sufficient to restore all metabolic differences.

Investigating the metabolic mechanisms underlying changes in cognitive function in a microgravity environment is of great scientific and social significance. Our research suggests that long-term orbital flights may lead to reversible brain damage at the metabolic level. This may lay a technological foundation for safeguarding brain health and provide new ideas and methods for the prevention and treatment of brain damage caused by microgravity.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1. Additional file 1: Materials and Methods. Table S1. Figure S1. (DOCX 8.9 MB)

Acknowledgements

The authors are grateful to all those who participated in this research for their contributions.

Author contributions

WL, XZ and XX designed the experiments. YL, XZ and WL wrote the manuscript. XZ, XC, BZ, JP and GK conducted the rhesus monkey experiments. XZ, XC, ZH, BZ and GK collected brain samples. YL, ZX, ZH, QZ and CZ performed mass spectrometry experiment. ZH, CL, ZY, ZW, ZL and GH conducted data analysis. All authors approved the final manuscript.

Funding

This work was supported by National Key Research and Development Program of China (2018YFE0126700), Shanghai Education Commission Research and Innovation Program (2019-01-07-00-02-E00037), Shanghai Municipal Commission of Science and Technology Program (21dz2210100, 2021SHZDZX), the “111” Program of Higher Education Discipline Innovation, China Postdoctoral Science Foundation (2021M702137) and Natural Science Foundation of Chongqing (cstc2021jcyj-msxmX1176, 2022NSCQ-MSX1304).

Data availability

All data generated or analyzed during this study are included in this article and Additional file1.

Declarations

Ethics approval

All procedures were performed in accordance with the principles of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), approved by Institutional Animal Care and Use Committee of China Astronaut Research and Training Center (ACC-IACUC-2014-001) and Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (IACUC-20140710).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Abbreviations

HDBR head-down bed rest

dmPFC dorsomedial prefrontal cortex

LC-MS liquid chromatography-mass spectrometry

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yuting Li and Xu Zhang contributed equally to this work.
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References

1. Zhang X et al. Simulated weightlessness procedure, head-down bed rest impairs adult neurogenesis in the hippocampus of rhesus macaque. Mol Brain 12 (2019).
2. Sun S, Yu HB, Yu RJ, Wang S. Functional connectivity between the amygdala and prefrontal cortex underlies processing of emotion ambiguity. Transl Psychiat 13 (2023).
3. Garrett-Bakelman FE et al. The NASA Twins Study: a multidimensional analysis of a year-long human spaceflight. Science 364 (2019).
4. Hargens AR Vico L Long-duration bed rest as an analog to microgravity J Appl Physiol 2016 120 891 903 10.1152/japplphysiol.00935.2015 26893033
Hargens AR, Vico L. Long-duration bed rest as an analog to microgravity. J Appl Physiol. 2016;120:891–903.26893033 10.1152/japplphysiol.00935.2015
5. Desai RI Kangas BD Limoli CL Nonhuman primate models in the study of spaceflight stressors: past contributions and future directions Life Sci Space Res 2021 30 9 23 10.1016/j.lssr.2021.03.008
Desai RI, Kangas BD, Limoli CL. Nonhuman primate models in the study of spaceflight stressors: past contributions and future directions. Life Sci Space Res. 2021;30:9–23.10.1016/j.lssr.2021.03.008
6. Wang C Targeting PDK2 rescues stress-induced impaired brain energy metabolism Mol Psychiatry 2023 28 4138 50 10.1038/s41380-023-02098-9 37188779
Wang C, et al. Targeting PDK2 rescues stress-induced impaired brain energy metabolism. Mol Psychiatry. 2023;28:4138–50.37188779 10.1038/s41380-023-02098-9
7. Ji G The mitochondrial proteomic changes of rat hippocampus induced by 28-day simulated microgravity PLoS ONE 2022 17 e0265108 10.1371/journal.pone.0265108 35271667
Ji G, et al. The mitochondrial proteomic changes of rat hippocampus induced by 28-day simulated microgravity. PLoS ONE. 2022;17:e0265108.35271667 10.1371/journal.pone.0265108
8. Xu T Liquid chromatography-mass spectrometry-based urinary metabolomics study on a rat model of simulated microgravity-induced depression J Pharm Biomed Anal 2019 165 31 40 10.1016/j.jpba.2018.11.058 30502550
Xu T, et al. Liquid chromatography-mass spectrometry-based urinary metabolomics study on a rat model of simulated microgravity-induced depression. J Pharm Biomed Anal. 2019;165:31–40.30502550 10.1016/j.jpba.2018.11.058
9. Raber J Effects of 5-Ion Beam Irradiation and Hindlimb unloading on metabolic pathways in plasma and brain of behaviorally tested WAG/Rij rats Front Physiol 2021 12 746509 10.3389/fphys.2021.746509 34646164
Raber J, et al. Effects of 5-Ion Beam Irradiation and Hindlimb unloading on metabolic pathways in plasma and brain of behaviorally tested WAG/Rij rats. Front Physiol. 2021;12:746509.34646164 10.3389/fphys.2021.746509
10. Genchi G An overview on D-amino acids Amino Acids 2017 49 1521 33 10.1007/s00726-017-2459-5 28681245
Genchi G. An overview on D-amino acids. Amino Acids. 2017;49:1521–33.28681245 10.1007/s00726-017-2459-5
11. Rosso IM Crowley DJ Silveri MM Rauch SL Jensen JE Hippocampus glutamate and N-Acetyl aspartate markers of excitotoxic neuronal compromise in posttraumatic stress disorder Neuropsychopharmacology 2017 42 1698 705 10.1038/npp.2017.32 28195577
Rosso IM, Crowley DJ, Silveri MM, Rauch SL, Jensen JE. Hippocampus glutamate and N-Acetyl aspartate markers of excitotoxic neuronal compromise in posttraumatic stress disorder. Neuropsychopharmacology. 2017;42:1698–705.28195577 10.1038/npp.2017.32
12. Shang XL Neural oscillations as a bridge between glutamatergic system and emotional behaviors in simulated microgravity-induced mice Behav Brain Res 2017 317 286 91 10.1016/j.bbr.2016.09.063 27693609
Shang XL, et al. Neural oscillations as a bridge between glutamatergic system and emotional behaviors in simulated microgravity-induced mice. Behav Brain Res. 2017;317:286–91.27693609 10.1016/j.bbr.2016.09.063
13. Van Ast VA Time-dependent effects of cortisol on the contextualization of emotional memories Biol Psychiatry 2013 74 11 809 16 10.1016/j.biopsych.2013.06.022 23972529
Van Ast VA, et al. Time-dependent effects of cortisol on the contextualization of emotional memories. Biol Psychiatry. 2013;74(11):809–16.23972529 10.1016/j.biopsych.2013.06.022
14. Quirarte GL Galvez R Roozendaal B McGaugh JL Norepinephrine release in the amygdala in response to footshock and opioid peptidergic drugs Brain Res 1998 808 134 40 10.1016/S0006-8993(98)00795-1 9767150
Quirarte GL, Galvez R, Roozendaal B, McGaugh JL. Norepinephrine release in the amygdala in response to footshock and opioid peptidergic drugs. Brain Res. 1998;808:134–40.9767150 10.1016/S0006-8993(98)00795-1
15. Koizumi S UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis Nature 2007 446 1091 5 10.1038/nature05704 17410128
Koizumi S, et al. UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. Nature. 2007;446:1091–5.17410128 10.1038/nature05704
