CoSi-500 Study - Confinement for 500 days - Evaluation of Stress and Immunity
  1. 2009 • Mars105
  2. 2010 • Mars500
Life Sciences:
  • Immunology and Haematology
  • Neuroscience
  • Psych-physiology (stress)
Jennifer Ngo-Anh
A. Choukèr (1), I. Kaufmann (1), S. Baatout (2), B. Morukov (3), T. Schachtner (1), G. Schelling (1), S. Kreth (1), O. Ullrich (4), S. Praun (5), M. Thiel (1)
Department of Anaesthesiology
Klinikum Großhadern
Marchioninistrasse 15
81377 München
Lab of Molecular & Cellular Biology
Belgian Nuclear Research Center, SCK-CEN
Boeretang 200
2400 Mol
State Research Center of The Russian Federation
Institute for Biomedical Problems (IBMP)
Khoroshevskoye Shosse 76 A
Moscow 123007
University of Zurich
Medical Faculty
Winterthurer Strasse 190
8057 Zurich
V&F medical development GmbH
Andreas-Hofer-Strasse 15
6067 Absam

The consequences of confinement and stress-associated neuroendocrine and immune changes were investigated for different duration, e.g. 10 days [SHIMAMIYA, T. et al. 2004], to 60 days [HENNIG, J. et al. 1996], as well as 110 days and 240 days [CHOUKER, A. et al. 2002]. Even though a full comparability of the studies mentioned above is not possible due to various study protocols, these studies revealed that confinement induces changes in neuroendocrine and immune responsiveness. Moreover, more specific information on changes of the human immune system have been described very recently from a month-long study in the Canadian arctic [CRUCIAN, B. et al. 2007] altogether indicating changes as also observed in astronauts after space flight, including T-cell depression. T-cell dysfunction was mediated by changes within the peripheral blood mononuclear cell compartment, including a paradoxical atypical monocytosis associated with altered production of inflammatory cytokines. In accordance to observation from space flight associated stressful situations that resulted in sympathetic and/or glucocorticoid-mediated immune down regulation [TINGATE, T. R. et al. 1997], and virus reactivation [STOWE, R. P. et al. 2000] [STOWE, R. P. et al. 2001] [STOWE, R. P. et al. 2003], we could also investigate the dynamics and severity of immune (dys-)function in long-term as well as short-tem approaches [CHOUKER, A. et al. 2002], [CHOUKER, A. et al. 2001]. We have shown that stressful conditions of psychological or physical nature can activate and/or paralyze specific immune responses, respectively. From these recent investigations we could confirm the role of the glucocorticoid- and catecholaminergic system together with stress scores as markers of physiologic stress responses. These stress dependent mechanisms can potentially result in an immune down regulation of by activation of the adenylylcyclase to catalyze the production of cAMP as been triggered by catecholamines [BOXER, L. A. et al. 1980, KOCH, T. et al. 1996] or other stress-systems (e.g. endocannabinoids) through the regulation by the same signal second messenger [KLEIN, T. W. et al. 2003], [A. CHOUKÈR, SCHELLING G et al, Neuroscience meeting, San Diego, 2007 abstract]. Those cAMP dependent mechanisms can impact a variety of immune cell functions in humans and animals and can modulate T-helper cell development and chemotaxis. In addition to these stress-associated effects, conditions of local or systemic inflammation, e.g. in the course of an injury/wound infection or general disease, respectively, may suppress immune responses by metabolites released from tissue and immune cells under these conditions. One of these endogenous metabolites is adenosine, released from direct tissue damage and by degradation of purine nucleotides [THIEL, M. et al. 2003] [DECKING, U. K. et al. 1997]. This increase also occurs under general normoxia (please see for detail the proposal submitted for CONCORDIA) in the course of  inflammation ([CHOUKER, A. et al. 2005] and KAUFMANN et al, ASA 2007, abstract). Because the effector function of adenosine is also triggered through the cAMP-coupled A2 receptors (R), (reviewed in [LINDEN, J. 2006]) it can down-regulate immune responses during local or general inflammation and likely potentiate the permissive activation of the catecholaminergic and the endocannabinoid-system via the same signal pathway.

In an integrated concept of earth bound and space-flight protocols we seek for a deeper insight and mechanistic understanding of the consequences of complex environmental factors on psycho-neuro-endocrine on the immune responses with special focus on the regulation of the hosts´ defence mechanisms (e.g. against bacteria). 

Long-term objectives:
The understanding of the complex interactions of stress and immunity might enable adequate health and immune monitoring and might as well suggest suitable countermeasures for the prevention of the unwanted immunological effects during long-term confinement and space mission.

The Mars500 isolation study was conducted at the Institute for Biomedical Problems (IBMP) in Moscow. It was run from 3 June 2010 to 4 November 2011, lasting 520 days.

Research design and methods include a monthly performance as conducted in our previous confinement studies. A defined set of parameters for hormonal, immunologic, metabolic and stress assessment will be determined from blood, saliva and urine samples as well as from questionnaires. In addition, newly validated stress and immune associated variables (e.g. endocannabinoids) and non-invasive techniques (exhaled breath analyses by Ion Molecule Reaction Mass-Spectrometry), will be included. Moreover, this monitoring might be applied (entirely or partly) also when a subject feels sick to allow as well assessment of acute changes which will help to further understand and track the patho-physiological adaptation of humans exposed to this extreme environmental condition. In addition, we intend to use Diffusion Tensor Imaging (DTI, facultative), a non x-ray imaging technique based on measurements of water diffusion in the brain. DTI by magnetic resonance imaging gives information on brain tissue microstructure in-vivo and allows the delineation of chronic stress effects on brain microcircuity. 

The understanding of the complex interactions of stress and immunity might enable adequate health and immune monitoring and might as well suggest suitable countermeasures for the prevention of the unwanted immunological effects during long-term confinement and space mission.


The psycho-neuroendocrine response is determined:

i) by the acute and chronic stress responses repeatedly determined by standardized questionnaires,
ii) by the neuroendocrine (stress-) responses by means of blood cannabinoids, saliva cortisol and urine catecholamines.

Moreover, beside the role of e.g. cortocoids to control immune functions, recent findings have also emphasized the role of nerval pathways to control the host´s immune response [TRACEY, K. J. 2002]. In this respect, pre- and post-confinement morphometric changes of brain areas affected by the conditions of chronic stress will be quantified by Diffusion Tensor Imaging (DTI) and Voxel-Based Volumetry using Magnetic Resonance Imaging (MRI) and interpreted in relation to neuroendocrine findings (facultative).  

The expected immunomodulation will hence be assessed by enumerative and functional, antigen-targeted ex vivo assays to detect subsequent changes of the innate and specific immune system. Furthermore, cell-surface receptor expression and cell signalling properties of immune cells will be evaluated on the basis of functional and molecular biology assays. E.g., to assess microbicidial and cytotoxic functions of the non-specific immune system, phagocytes will be challenged with particulate stimuli and their adhesion, phagocytosis and the associated bactericidal respiratory burst activity as well as the H2O2 production following soluble stimuli will be measured [KAUFMANN, I. et al. 2006]. To obtain functions of the specific part of the immune system, antigen-specific immune responses to viral, fungal and bacterial antigens will be determined providing insight into Th1/Th2/Th3 weighed responses, also by intracellular cytokine staining, extra-cellular cytokines secretion and on the molecular level.

Moreover, the cells´ metabolic stress being affected through infections/inflammation will be monitored by the quantification of purine (e.g. adenosine) blood concentrations, which exert important hormone-like immune-modulation. 
The consequences of changes in the balance of immune activation and suppression are known from clinical trials to result in particular cell damage and oxidative stress as a consequence of immune dysfunction. In that course, uncontrolled activation of the innate immunity will induce cell´s oxidative and catabolic inflammatory stress-responses that can be determined in blood. Interestingly, most of these substances are volatile and can be quantified in exhaled air by Ion Molecule Reaction Quadrupol mass-spectrometry (IMR-MS (method as described in [POTYK, D. K. et al. 1998]).  
Altogether, the selection of parameters for the determination of psychoneuroendocrine, immune and cell´s stress parameters as described as above and in the tables of the Addendum include on the one hand i) a defined set of variables that has been used in previous and ongoing studies and on the other hand ii) is completed by new and innovative parameters and techniques to allow an even more differentiated and somewhat non-invasive immune monitoring. This approach will hereby allow to track the patho- physiological adaptaption of humans exposed to this extreme, unique environmental condition by using a variety of standardized but also new variables and will guarantee that knowledge achieved can be subjected to one-to-one comparisons of results gathered from previous, current and future “space-related” studies and also to clinical investigations, where the same set of data has been achieved under different conditions of disease (e.g. in the course of stress associated disease or during inflammation). This comparison of data sets will enable a better estimation of the pathogenetic value and clinical impact for health of those parameters collected and analyzed during e.g. MARS500 and will hereby significantly increase the scientific value our investigations.

Figure 1: Interaction of psycho-neuroendocrine responses, the immune-modulation and the (cell) metabolism. For each topic various established and also newly validated parameters and methods will be applied.

Finally, one major added value is likely to be obtained by the comparison of the new results after 100 (control, test-phase) and 500 days of confinement with those measured in our previous studies and protocols planned for future investigation. Thus, this protocol is designed to allow for comparison of results of several earth-bound and space flight control groups. This is achievable due to the assessment of a solid “rack-set” of parameters in former and ongoing scientific studies: 

i) “historical” and potential “future” earth-bound confinement controls of studies with the  participation of our research groups e.g. confinement for 110/240 or Antarctica
ii) current investigation on the effects of (stress-associated) immune changes on the International Space Station (esa-IMMUNO-project). 

iii) current clinical investigations on the role of psychoneuroendocrine and immune changes in sub-acute and chronic stress models in patients, e.g. during surgical stress [SCHELLING, G. et al. 2006], during intensive care therapy [Schelling et al, in preparation] as well as in patients suffering from chronic stress and pain [KAUFMANN I et al., 2007 in revision], respectively. 

Specific comments on the parameters and samples 

Saliva, Blood, Urine and Paper Test analyses 

The panel for the assessment of psychoneuroendocrine and immune changes includes parameters that are determined from saliva, blood and urine and from paper tests (please see in detail in the Addendum).  
In addition and beside blood collections as described above, the non-invasive technique of breath analyses by Ion Molecule Reaction (IMR)-Mass-Spectrometry (MS) represents a scientifically attractive and complementary breakthrough for longitudinal monitoring.

Molecular Breath Gas Analyses (MBGA) 

In the recent decade it has emerged that many different volatile organic compounds (VOC) are present in the exhaled breath and that some of these compounds can serve as indicators for multiple physiologic functions and a considerable number of disease states ranging from metabolic disorders (e.g. diabetes) to pulmonary disease (e.g. asthma), infections and cancer.

The characterization of biomarkers in exhaled breath using Molecular Breath Gas Analyses (MBGA) by Ion Molecule Reaction Mass Spectrometry (IMR-MS) is a promising technique which results from research activities including clinical studies conducted by our group and partners (The overall increasing interest of the scientific community towards this new research field has resulted in the last years in the Foundation of The International Association for Breath Research (IABR) and the Journal of Breath Research). 
The molecular breath gas analyses by mass-spectrometry will be conducted in order to determine biological markers and bio-products in exhaled air. The molecular analyses of exhaled breath offer a novel and noninvasive method to diagnose a variety of organ dysfunctions including metabolic changes on the basis of the determination of more than 240 masses by IMR-MS. This method will be important to allow the quantification of: 

i) the changes in immune activity and oxidative stress as reflected by changes in nitric oxide, proprionic acid, isoprene, pentane and malondialdehyde concentrations. Moreover,  

ii) very recent and unpublished data indicate that specific compounds of different masses are potentially applicable to indirectly follow up the release of stress hormone-like substances from the metabolism and  

iii) likewise, MBGA may also monitor humans´ glucose and fat metabolism, both of interest in the understanding of the consequences of long-term hypoxia. The results obtained from MBGA will be interpreted in conjunction with the above mentioned sets of blood, urine and saliva analyses that will be compared in parallel on-site. 

The IMR-MS has now been applied successfully for clinical research purposes during the last 2 years and proven as a non-invasive and highly reliable tool to monitor compounds in the blood of patients [HORNUSS, C. et al. 2007]. This IMR-MS technique is also highly sensitive to monitoring inflammation [Dolch, Schelling, manuscript submitted] as well as oxidative and metabolic changes due to physical and oxidative stress (ref to add?). Therefore, the IMR-MS will help to monitor immunological changes (e.g. parameters of cell damage and changes in cell oxidative stress, release of NO, and other oxidative/metabolic stress parameters as also recently been characterized during short-term confinement (in collaboration with Dr. Heer, DLR, Cologne, Germany) and in samples collected during the 8th and 9th DLR Parabolic Flight campaign (May and November 2006, [CHOUKÈR A; International Association for Breath Research IABR-meeting abstract, Cleveland 2007, USA]). In these studies for instance, exhaled propionaldehyde concentrations were paralleled by functional changes of granulocytes (oxygen radical production). Taken together, IMR-MS (method as described by [HORNUSS, C. et al. 2007]) will unambiguously help to identify variables with great significance for health assessment and monitoring.

Diffusion Tensor Imaging (DTI) 

Moreover and facultative, the use of the recent brain DTI (Diffusion Tensor Imaging)-technique could be applied to investigate for the first time the consequences of confinement on stress associated structural changes in the brain. DTI is a recently emerged, variation of MRI which quantifies the diffusion of water in various tissues and allows the non-invasive visualization of tissue microstructure in-vivo. It is based upon the phenomenon of thermally driven water diffusion, known as Brownian motion.  This random motion of water molecules can be quantified and the parameters reflect intrinsic features of tissue microstructure. Tissues which have a random microstructure, such as cerebrospinal fluid (CSF), show isotropic diffusion properties [PAGLIARO, P. et al. 2001]. This means that diffusion is equal in all directions. In contrast, in tissues that have a regularly ordered microstructure, such as brain white matter; water molecules behave in a sorted fashion, with a predominant motion direction and a given orientation, hence indicating a marked anisotropy in the diffusion properties [PERALTA, C. et al. 2000]. The microstructural tissue changes can be expressed as fractional anisotropy (FA), which has no dimension and as apparent diffusion coefficient (ADC, mean diffusitivity) which is expressed as 10-6 mm2/s [INGLOTT, F. S. et al. 2000]. These indices can be seen as complementary for the evaluation of brain tissue. Whereas ADC is a measure of the directionally averaged magnitude of diffusional motion of water molecules (= related to integrity of membranes), FA describes the degree of anisotropy of the process of  molecular diffusion (= degree of structural alignment) [INGLOTT, F. S. et al. 2000]. ADC is thought to be homogenous throughout the different brain regions of grey and white matter [INGLOTT, F. S. et al. 2000]. Due to the directionality of the fiber organization, FA in contrast varies in its magnitude in different brain structures and tissue types. For example, FA value of the cerebrospinal fluid (CSF) is close to 0, whereas FA of the corpus callosum, in which neural fibres are arranged in a regular and parallel fashion, approaches 1.  
DTI is simple to perform, requires no contrast-media and is completed within 20 minutes. Imaging could be conducted once prior to the onset of confinement and a second time after end of confinement (in the first 2-4 weeks, if possible, imaging will be performed in Munich). As mentioned before, DTI is not a prerequisite for the performance of the study but could, however, give a completely new insight into adaptive changes in the brain as a functional morphometric correlation of chronic stress and inflammation. The newly appreciated neural anti-inflammatory pathway provides the clear rationale to study brain-immune interactions also on a morphometric basis in the brain. These new in vivo measurement will hence allow analyses of morphological changes in specific stress adaptive regions of the human brain [ABE, O. et al. 2006]. 
We used DTI in patients with chronic stress-related pathology to measure FA and ADC in brain areas regulating traumatic memory and stress response in a previous study. Higher values of FA/RA and lower values of ADC reflected increased complexity of brain tissue microstructure. Stress-related pathology including altered glucocorticoid activity, a high number of traumatic memories and chronic stress symptoms was  associated with changes in structural plasticity within the amygdala and the hippocampus in the adult brain [G. Schelling et al., Abstract “Society for Neuroscience”- meeting, USA Oct. 2006; full research manuscript submitted on basis of Fig. 2]. Figure 2 gives an example of brain areas with significantly different microstructures in patients with a stress-related disorder. 

Figure 2: Grey matter regions with significantly higher fractional anisotropy values (p<0.001) in 30 patients with stress-related disorders after a prolonged and severe stress exposure when compared to 30 healthy age and sex comparable controls (Schelling G. et al, 2007, manuscript in preparation).

Sample collection

According to the protocol in the previous confinement study (SFINCSS´99) a monthly (during the 30-35 day routine analyse cycles) assessment is advised to mirror the stress-associated neuro-endocrine, metabolic and immunological changes. In addition to these standardized time points to collect the respective samples, health assessment monitoring by this set of tests (entirely or partly) might be applied at the moment when the subject starts to feel sick to allow the determination of acute changes. The panel of investigations will include stress tests, blood (morning), saliva (morning and evening), urine (2 x 12 h) and air breath collection (morning and evening). 

We seek to include all subjects over the entire study period including two pre (e.g. -30, -7 days) and up to three post-time points (+1, +7, +14/30). Additionally, at the pre and post-time points the PTSS10 and Spielbergers´ test (see below in Addendum) will be conducted. However, this needs to be determined with the overall schedule and other scientific proposal and logistic needs. In addition to this standardized health assessment monitoring it is recommended, but not as a pre-requisite, to apply this rack of test (entirely or partly) also when the subject starts to feel sick to allow the determination of unexpected and acute changes.

Figure 3: Sample collection intervals for the MARS500-study: scenarios for 100 days (test) and 500 days of confinement. The set of parameters is determined by stress test, saliva, urine, blood and breathing air samples twice pre, every 30-35 days during 100 and 500 days of confinement and twice post-confinement, respectively (indicated by thin black arrows). It needs to be further determined, if sample assessment during 500 days confinement is possible during "Mars landing scenario" which should be included if possible. In addition, non-x-ray brain imaging might be conducted once pre and once post-confinement in Munich (when appropriate), as indicated by "(DTI)" and a grey-coloured arrows.


We hypothetize that

1.) extensive long-term confinement induces psycho-neuro-endocrine and metabolic cell stress responses which will all affect the human immune system with increasing intensity,

2.) monitoring of (patho-) physiological, stress dependent neuro-endocrine, metabolic and immune changes can be achieved by the help of improved techniques and technologies in minute amounts of blood or even non-invasively, and that

3.) a distinguished analysis of the complex man-environment interactions will enable, together with data from the ISS and several earth-bound control studies, future immune targeted countermeasures.

RESULTS for Brain DTI:
The participants of the MARS500 project underwent 3T-MR imaging including a dedicated DTI-sequence (Diffusion Tensor Imaging) before the isolation, right after ending of confinement and 6 months after the experiment. DTI models the diffusion of water molecules which is directed alongside natural boundaries, e.g. axonal fiber bundles. By evaluating different DTI-scalars, conclusions about the underlying anatomy can be drawn in a microstructural level.
As part of multiple physiological and clinical experiments conducted during MARS500, we chose Diffusion Tensor Imaging to test for possible effects of long term confinement on white matter integrity.
Voxelwise statistical analysis of the DTI data was carried out using tract-based-spatial statistics, comparing an age-matched control group.
At all three sessions, significant lower fractional anisotropy (FA) than in controls was found in the anterior parts of the callosal body of the participants. Furthermore, after ending of confinement a wide-spread FA reduction could be seen in the right hemisphere culminating in the temporo-parietal-junction-zone. All these areas with decreased FA predominantly showed an elevated radial diffusivity and mean diffusivity while axial diffusivity was less correlated.
Long-term confinement does have measurable effects on the microstructure of the brain white matter. We assume effects of sensory deprivation to account for the regional FA reductions seen in the right TPJ.
The differences in the Corpus callosum were interpreted as due to preliminary conditions, e.g. personality traits or training effects. FA and radial diffusivity were the predominant DTI parameters with significant changes, suggesting underlying processes of myelin plasticity.

for more details on the brain DTI results, please, consult Ref. Doc.: 
"Changes of brain DTI in healthy human subjects after 520 days isolation and confinement on a simulated mission to Mars", Life Sciences in Space Research24, https://doi.org/10.1016/j.lssr.2019.09.004, pp. 83-90.
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Figure 1: Interaction of psycho-neuroendocrine responses, the immune-modulation and the (cell) metabolism. For each topic various established and also newly validated parameters and methods will be applied.

Figure 2: Grey matter regions with significantly higher fractional anisotropy values (p<0.001) in 30 patients with stress-related disorders after a prolonged and severe stress exposure when compared to 30 healthy age and sex comparable controls (Schelling G. et al, 2007, manuscript in preparation).

Figure 3: Sample collection intervals for the MARS500-study: scenarios for 100 days (test) and 500 days of confinement. The set of parameters is determined by stress test, saliva, urine, blood and breathing air samples twice pre, every 30-35 days during 100 and 500 days of confinement and twice post-confinement, respectively (indicated by thin black arrows). It needs to be further determined, if sample assessment during 500 days confinement is possible during "Mars landing scenario" which should be included if possible. In addition, non-x-ray brain imaging might be conducted once pre and once post-confinement in Munich (when appropriate), as indicated by "(DTI)" and a grey-coloured arrows.

Air breathing samples from the Mars500 crew members. - Photo Credits: ESA/IMBP

Some of the more than thousand saliva samples from the Mars500 crew members. - Photo Credits: ESA/IMBP
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