Proteomic Analysis of Mouse Hypothalamus under Simulated Microgravity

Exposure to altered microgravity during space travel induces changes in the brain and these are reflected in many of the physical behavior seen in the astronauts. The vulnerability of the brain to microgravity stress has been reviewed and reported. Identifying microgravity-induced changes in the brain proteome may aid in understanding the impact of the microgravity environment on brain function. In our previous study we have reported changes in specific proteins under simulated microgravity in the hippocampus using proteomics approach. In the present study the profiling of the hypothalamus region in the brain was studied as a step towards exploring the effect of microgravity in this region of the brain. Hypothalamus is the critical region in the brain that strictly controls the pituitary gland that in turn is responsible for the secretion of important hormones. Here we report a 2-dimensional gel electrophoretic analysis of the mouse hypothalamus in response to simulated microgravity. Lowered glutathione and differences in abundance expression of seven proteins were detected in the hypothalamus of mice exposed to microgravity. These changes included decreased superoxide dismutase-2 (SOD-2) and increased malate dehydrogenase and peroxiredoxin-6, reflecting reduction of the antioxidant system in the hypothalamus. Taken together the results reported here indicate that oxidative imbalance occurred in the hypothalamus in response to simulated microgravity.     

Evaluation of gene, protein and neurotrophin expression in the brain of mice exposed to space environment for 91 days

Effects of 3-month exposure to microgravity environment on the expression of genes and proteins in mouse brain were studied. Moreover, responses of neurobiological parameters, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), were also evaluated in the cerebellum, hippocampus, cortex, and adrenal glands. Spaceflight-related changes in gene and protein expression were observed. Biological processes of the up-regulated genes were related to the immune response, metabolic process, and/or inflammatory response. Changes of cellular components involving in microsome and vesicular fraction were also noted. Molecular function categories were related to various enzyme activities. The biological processes in the down-regulated genes were related to various metabolic and catabolic processes. Cellular components were related to cytoplasm and mitochondrion. The down-regulated molecular functions were related to catalytic and oxidoreductase activities. Up-regulation of 28 proteins was seen following spaceflight vs. those in ground control. These proteins were related to mitochondrial metabolism, synthesis and hydrolysis of ATP, calcium/calmodulin metabolism, nervous system, and transport of proteins and/or amino acids. Down-regulated proteins were related to mitochondrial metabolism. Expression of NGF in hippocampus, cortex, and adrenal gland of wild type animal tended to decrease following spaceflight. As for pleiotrophin transgenic mice, spaceflight-related reduction of NGF occurred only in adrenal gland. Consistent trends between various portions of brain and adrenal gland were not observed in the responses of BDNF to spaceflight. Although exposure to real microgravity influenced the expression of a number of genes and proteins in the brain that have been shown to be involved in a wide spectrum of biological function, it is still unclear how the functional properties of brain were influenced by 3-month exposure to microgravity.     

Effect of Simulated Microgravity on Human Brain Gray Matter and White Matter – Evidence from MRI

Background

There is limited and inconclusive evidence that space environment, especially microgravity condition, may affect microstructure of human brain. This experiment hypothesized that there would be modifications in gray matter (GM) and white matter (WM) of the brain due to microgravity.

Method

Eighteen male volunteers were recruited and fourteen volunteers underwent -6° head-down bed rest (HDBR) for 30 days simulated microgravity. High-resolution brain anatomical imaging data and diffusion tensor imaging images were collected on a 3T MR system before and after HDBR. We applied voxel-based morphometry and tract-based spatial statistics analysis to investigate the structural changes in GM and WM of brain.

Results

We observed significant decreases of GM volume in the bilateral frontal lobes, temporal poles, parahippocampal gyrus, insula and right hippocampus, and increases of GM volume in the vermis, bilateral paracentral lobule, right precuneus gyrus, left precentral gyrus and left postcentral gyrus after HDBR. Fractional anisotropy (FA) changes were also observed in multiple WM tracts.

Conclusion

These regions showing GM changes are closely associated with the functional domains of performance, locomotion, learning, memory and coordination. Regional WM alterations may be related to brain function decline and adaption. Our findings provide the neuroanatomical evidence of brain dysfunction or plasticity in microgravity condition and a deeper insight into the cerebral mechanisms in microgravity condition.     

Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the nasa rotating wall vessel bioreactor

Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.     

Protein pattern of Xenopus laevis embryos grown in simulated microgravity

Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.     

Default network connectivity decodes brain states with simulated microgravity

With great progress of space navigation technology, it becomes possible to travel beyond Earth’s gravity. So far, it remains unclear whether the human brain can function normally within an environment of microgravity and confinement. Particularly, it is a challenge to figure out some neuroimaging-based markers for rapid screening diagnosis of disrupted brain function in microgravity environment. In this study, a 7-day ?6° head down tilt bed rest experiment was used to simulate the microgravity, and twenty healthy male participants underwent resting-state functional magnetic resonance imaging scans at baseline and after the simulated microgravity experiment. We used a multivariate pattern analysis approach to distinguish the brain states with simulated microgravity from normal gravity based on the functional connectivity within the default network, resulting in an accuracy of no less than 85 % via cross-validation. Moreover, most discriminative functional connections were mainly located between the limbic system and cortical areas and were enhanced after simulated microgravity, implying a self-adaption or compensatory enhancement to fulfill the need of complex demand in spatial navigation and motor control functions in microgravity environment. Overall, the findings suggest that the brain states in microgravity are likely different from those in normal gravity and that brain connectome could act as a biomarker to indicate the brain state in microgravity.     

Effect of 2400 MHz mobile phone radiation exposure on the behavior and hippocampus morphology in Swiss mouse model

Electromagnetic field exposure to the nervous system can cause neurological changes. The effects of extremely low-frequency electromagnetic fields, such as second-generation and third-generation radiation, have been studied in most studies. The current study aimed to explore fourth-generation cellular phone radiation on hippocampal morphology and behavior in mice. Swiss albino male mice (n = 30) were randomly categorized into 3 groups; control, 40 min, and 60 min exposure to 2400 MHz radiofrequency electromagnetic radiation (RF-EMR) daily for 60 days. The control mice were housed in the same environments but were not exposed to anything. Anxiety-like behaviors were tested using the elevated plus-maze. For histological and stereological examination, the brain was dissected from the cranial cavity. On Cresyl violet stained brain slices, the number of pyramidal neurons in the cornu ammonis of the hippocampus were counted. In exposed mice compared to control mice, a significant increase in anxiety-like behavior has been observed. Histological observations have shown many black and dark blue cytoplasmic cells with shrunken morphology degenerative alterations in the neuronal hippocampus in the radiation exposed mice. In the RF-EMR mouse hippocampus, stereological analyses revealed a significant decrease in pyramidal and granule neurons compared to controls. Our findings suggest that 2400-MHz RF-EMR cell phone radiation affects the structural integrity of the hippocampus, which would lead to behavioral changes such as anxiety. However, it alerts us to the possible long-term detrimental effects of exposure to RF-EMR.     

Altered Baseline Brain Activity with 72 h of Simulated Microgravity – Initial Evidence from Resting-State fMRI

To provide the basis and reference to further insights into the neural activity of the human brain in a microgravity environment, we discuss the amplitude changes of low-frequency brain activity fluctuations using a simulated microgravity model. Twelve male participants between 24 and 31 years old received resting-state fMRI scans in both a normal condition and after 72 hours in a −6° head down tilt (HDT). A paired sample t-test was used to test the amplitude differences of low-frequency brain activity fluctuations between these two conditions. With 72 hours in a −6° HDT, the participants showed a decreased amplitude of low-frequency fluctuations in the left thalamus compared with the normal condition (a combined threshold of P<0.005 and a minimum cluster size of 351 mm³ (13 voxels), which corresponded with the corrected threshold of P<0.05 determined by AlphaSim). Our findings indicate that a gravity change-induced redistribution of body fluid may disrupt the function of the left thalamus in the resting state, which may contribute to reduced motor control abilities and multiple executive functions in astronauts in a microgravity environment.     

Effects of simulated microgravity on the expression of presynaptic proteins distorting the GABA/glutamate equilibrium – A proteomics approach

Microgravity may cause cognition‐related changes in the animal nervous system due to the resulting uneven flow of fluids in the body. These changes may restrict the long‐term stay of humans in space for various purposes. In this study, a rat tail suspension model (30^o) was used to explore the effects of 21 days of prolonged simulated microgravity (SM) on the expression of proteins involved in cognitive functions in the rat hippocampus. SM decreased the content of γ‐aminobutyric acid (GABA) and increased the content of glutamate (Glu) in the rat hippocampus. A comparative ¹⁸O‐labeled quantitative proteomics strategy was applied to detect the differential expression of synaptic proteins under SM. Fifty‐three proteins were found to be differentially expressed under SM. Microgravity induces difficulty in the formation of the SNARE complex due to the down‐regulation of vesicle‐associated membrane protein 3(VAMP3) and syntaxin‐1A. Synaptic vesicle recycling may also be affected due to the dysregulation of syntaxin‐binding protein 5 (tomosyn), rab3A and its effector rim2. Both processes are disturbed, indicating that presynaptic proteins mediate a GABA/Glu imbalance under SM. These findings provide clues for understanding the mechanism of the GABA/Glu equilibrium in the hippocampus induced by microgravity in space and represent steps toward safe space travel.     

模拟失重对白假丝酵母菌增殖和毒性的影响

【背景】近年来研究发现,失重条件可对一些致病微生物的增殖和毒性产生影响,白假丝酵母菌(Candida albicans)是典型的条件性致病真菌,在太空环境和人体中普遍存在,研究失重条件下白假丝酵母菌的增殖和毒性意义重大。【目的】利用旋转细胞培养系统(Rotary cell culture system,RCCS)模拟失重环境对白假丝酵母菌进行连续传代培养,检测模拟失重环境对白假丝酵母菌增殖情况、毒性以及基因表达的变化。【方法】将白假丝酵母菌接种在旋转生物反应器(High aspect rotating vessel,HARV)中,利用旋转细胞培养系统连续传代培养14 d,然后对菌株进行增殖速率测定、不同pH条件下增殖能力测定、生物膜相对形成能力测定和细胞毒性和动物毒力测定;利用转录组测序技术找出差异表达基因,结合性状分析模拟失重可能对白假丝酵母菌增殖和毒力的影响。【结果】与对照组相比,模拟失重组白假丝酵母菌对数期提前,增殖速率加快,在适宜pH条件下的增殖能力普遍提高,但其生物膜形成能力相对减弱,对LoVo细胞和小鼠的毒性减弱;转录组测序发现,模拟失重组共有280个基因表达差异达1.5倍以上(P0.05),其中248个上调、32个下调。差异基因经基因功能注释(Gene ontology,GO)和京都基因及基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析发现,相关胞膜形成及细胞分裂基因表达上调,生物膜形成、细胞黏附及共生粘连宿主基因表达下调。【结论】模拟失重环境可引起白假丝酵母菌增殖和毒性水平发生变化,相关改变可为研究失重环境对微生物的影响提供参考。     

Surface transformation of bioactive glass in bioreactors simulating microgravity conditions. Part I: experimental study

Surface modified bioactive glass with surface properties akin to those of the bone mineral phase is an attractive candidate for use as a microcarrier material for 3-D growth of bone-like tissue in rotating wall vessel bioreactors (RWVs). The critical surface properties of this material are the result of reaction in solution. Because an RWV environment is completely different from conditions previously employed for bioactive glass testing, a detailed study of the surface reactions is warranted. Under properly chosen conditions, RWVs can also provide a simulated microgravity environment for the bioactive glass (BG) particles. In this sense, this study is also a report on the behavior of a bioactive material under microgravity conditions simulated on earth. A high aspect ratio vessel (HARV) and carefully selected experimental conditions enabled the simulation of microgravity in our laboratory. A complimentary numerical study was simultaneously conducted to ascertain the appropriateness of the experimental parameters (particle size, particle density, medium density, medium viscosity, and rotational speed) that ensure simulated microgravity conditions for the glass particles in the HARV. Physiological solutions (pH 7.4) with and without electrolytes, and also with serum proteins, were used to study the change in surface character resulting from simulated microgravity. Control tests at normal gravity, both static and dynamic, were also conducted. Solution and surface analyses revealed major effects of simulated microgravity. The rates of leaching of constituent ions (Si-, Ca-, and P-ions) were greatly increased in all solutions tested. The enhanced dissolution was followed by the enhanced formation of bone-like minerals at the BG surface. This enhancement is expected to affect adsorption of serum proteins and attachment molecules, which, in turn, may favorably affect bone cell adhesion and function. The findings of the study are important for the use of bioactive materials as microcarriers to generate and analyze 3-D bone-like tissue structures in bioreactors under microgravity conditions or otherwise. Copyright John Wiley & Sons, Inc.     

Neurobiology of Mice Selected for High Voluntary Wheel-running Activity

Selective breeding of house mice has been used to study theevolution of locomotor behavior. Our model consists of 4 replicatelines selectively bred for high voluntary wheel running (High-Runner)and 4 bred randomly (Control). The major changes in High-Runnerlines appear to have taken place in the brain rather than incapacities for exercise. Their neurobiological profile resemblesfeatures of human Attention Deficit Hyperactivity Disorder (ADHD)and is also consistent with high motivation for exercise asa natural reward. Both ADHD and motivation for natural rewards(such as food and sex), as well as drugs of abuse, have beenassociated with alterations in function of the neuromodulatordopamine, and High-Runner mice respond differently to dopaminedrugs. In particular, drugs that block the dopamine transporterprotein (such as Ritalin and cocaine) reduce the high-intensityrunning of High-Runner mice but have little effect on Controlmice. In preliminary studies of mice exercised on a treadmill,brain dopamine concentrations did not differ, suggesting thatchanges in the dopamine system may have occurred downstreamof dopamine production (e.g., receptor expression or transduction).Brain imaging by immunohistochemical detection of c-Fos identifiedseveral key regions (prefrontal cortex, nucleus accumbens, caudate-putamen,lateral hypothalamus) that appear to play a role in the differentialresponse to Ritalin and in the increased motivation for runningin High-Runner mice. The activation of other brain regions,such as the hippocampus, was closely associated with wheel runningitself. Chronic wheel running (several weeks) also increasedthe production of new neurons to apparently maximal levels inthe hippocampus, but impaired learning in High-Runner mice.We discuss the biomedical implications of these findings.     

miR‐181c‐5p mediates simulated microgravity‐induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase

Impaired osteoblast proliferation plays fundamental roles in microgravity‐induced bone loss, and cell cycle imbalance may result in abnormal osteoblast proliferation. However, whether microgravity exerts an influence on the cell cycle in osteoblasts or what mechanisms may underlie such an effect remains to be fully elucidated. Herein, we confirmed that simulated microgravity inhibits osteoblast proliferation. Then, we investigated the effect of mechanical unloading on the osteoblast cell cycle and found that simulated microgravity arrested the osteoblast cell cycle in the G₂ phase. In addition, our data showed that cell cycle arrest in osteoblasts from simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR‐181c‐5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3′UTR. Lastly, we demonstrated that inhibition of miR‐181c‐5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G₂ phase in primary mouse osteoblasts partially through the miR‐181c‐5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity‐induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading.     

Changes in myocardial contractility and contractile proteins after four weeks of simulated [correction of simulate] weightlessness in rats

The interaction between the gravitational field, the position of the body, and the functional characteristics of the blood vessels determines the distribution of intravascular volume. In turn, this distribution determines cardiac pump function. One of the most profound circulatory changes that occurs in man during exposure to weightlessness is a cephalad redistribution of fluid caused by the lack of hydrostatic pressure in this microgravitative environment. The cephalad redistribution of fluid results in a loss of blood volume and then induces a decrease in preload. Recently, a decrease in sensitivity of arteriole to catecholamine has reported in rats of simulated weightlessness. This change in arteriole may reduce afterload. As a result, cardiovascular system may be shifted to a hypokinetic state during weightlessness condition for long-term. Echocardiographic data from astronauts during space flight showed an increase in heart rate, a 12 % decrease in stroke volume, and a 16 % decrease in left end diastolic volume. Electron-microscopic studies have shown changes in cardiac morphology in rats after exposure to microgravity for 7-12.5 days. After the COSMOS 2044 flight for 14 days, the light-microscopic studies have shown an atrophy of papillary muscles in rats left cardiac ventricle. It is not clear whether the function of atrophic myocardium is impaired. The data in three aspects as mentioned above suggest that weightlessness or simulated weightlessness may decrease the myocardial function. However, definite changes in cardiac performance have been hard to prove due to many limits. This studies were to answer two questions: Is the myocardial contractility depressed in rats subjected to simulated weightlessness for four weeks? What are the underlying mechanisms of the changing contractility?     

Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells

OBJECTIVES: Microgravity is known to affect the differentiation of bone marrow mesenchymal stem cells (BMSCs). However, a few controversial findings have recently been reported with respect to the effects of microgravity on BMSC proliferation. Thus, we investigated the effects of simulated microgravity on rat BMSC (rBMSC) proliferation and their osteogeneic potential. MATERIALS AND METHODS: rBMSCs isolated from marrow using our established effective method, based on erythrocyte lysis, were identified by their surface markers and their proliferation characteristics under normal conditions. Then, they were cultured in a clinostat to simulate microgravity, with or without growth factors, and in osteogenic medium. Subsequently, proliferation and cell cycle parameters were assessed using methylene blue staining and flow cytometry, respectively; gene expression was determined using Western blotting and microarray analysis. RESULTS: Simulated microgravity inhibited population growth of the rBMSCs, cells being arrested in the G(0)/G(1) phase of cell cycle. Growth factors, such as insulin-like growth factor-I, epidermal growth factor and basic fibroblastic growth factor, markedly stimulated rBMSC proliferation in normal gravity, but had only a slight effect in simulated microgravity. Akt and extracellular signal-related kinase 1/2 phosphorylation levels and the expression of core-binding factor alpha1 decreased after 3 days of clinorotation culture. Microarray and gene ontology analyses further confirmed that rBMSC proliferation and osteogenesis decreased under simulated microgravity. CONCLUSIONS: The above data suggest that simulated microgravity inhibits population growth of rBMSCs and their differentiation towards osteoblasts. These changes may be responsible for some of the physiological changes noted during spaceflight.     

14-3-3 expression in denervated hippocampus after entorhinal cortex lesion assessed by culture-derived isotope tags in quantitative proteomics

Activation of astrocytes accompanies many brain pathologies. Reactive astrocytes have a beneficial role in acute neurotrauma but later on might inhibit regeneration. 2D-gel electrophoresis and mass spectrometry were applied to study the proteome difference in denervated hippocampus in wildtype mice and mice lacking intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin (GFAP-/-Vim-/-) that show attenuated reactive gliosis and enhanced posttraumatic regeneration. Proteomic data and immunohistochemical analyses showed upregulation of the adapter protein 14-3-3 four days postlesion and suggested that 14-3-3 upregulation after injury is triggered by reactive gliosis. Culture-derived isotope tags (CDIT) and mass spectrometry demonstrated that 14-3-3 epsilon was the major isoform upregulated in denervated hippocampus and that its upregulation was attenuated in GFAP-/-Vim-/- mice and thus most likely connected to reactive gliosis.