A New Nutritional Countermeasure to Prevent the Deconditioning Induced by 60 Days of Antiorthostatic Bed Rest

This will be performed by using MR scanning sequences that specifically measure water and fat signals then calculate the respective content.

Next generation sequencing (NGS) of IgM heavy chain transcripts extracted from PBMC will 1) indicate if the expression frequencies of Ig gene segments are modified during bed rest thereby indicating a change in B lymphopoiesis (because each B cell created in the marrow is characterized by its own VH-D-JH-Cµ mRNA) and 2) allow the assessment of the activities of enzymes required for the formation of the B cell antigen receptor repertoire.

Flow cytometry will be used to determine if there is an egress of HSC/progenitors from the bone marrow into blood stream thereby indicating a decrease of the interactions between HSC and their niche. The same technique will be used to determine if there are changes in B cell subsets in peripheral blood.

Mass spectrometry will be performed on proteins obtained from muscle biopsies, microdialysis and serum. The generated peptides will be analysed using high resolution mass shot-gun mass spectrometry.

The end point is a reduction in muscle pump indices: a) decrease in causality between EMG and Blood pressure, b) decrease in gain between EMG and Blood pressure, and c) decrease in the interaction time between EMG and Blood pressure.

Skeletal muscle insulin resistance will be assessed during a euglycaemic hyperinsulinaemic clamp.

Whole blood will be immediately analyzed for glucose concentration, serum insulin will be quantified by radioimmunoassay and catecholamines will be quantified by high-performance liquid chromatography. Peripheral insulin sensitivity will be calculated during the final hour of the clamp in steady state.

Both structural and functional brain changes associated with head-down bedrest will be examined by 3T MRI. Imaging sequence parameters will be selected to maximize sensitivity to effects of interest.

Subjects perform a video game-like task of shooting a projectile out of a hand-held tool so that it passes between the gaps of an oriented target grill. Since the projectile and the tool are long and narrow, the subject must place the tool at the same orientation as the grill to achieve success. On certain trials, the subject is required to move the head between the time that the target is acquired and the moment of the response. Trials vary in terms of what the subject can see and feel during the execution of the task, and on body posture.

A 3D motion caption system is used to track in real-time the movements of the subject' head and hands. A virtual reality helmet is used to provide visual and audio information. A moving head support will be used to allow the subject to tilt the head laterally in both seated and laying conditions.

Change in pulse wave velocity in carotid and femoral arteries will be assessed by non-invasive ultrasound measurements.

Within-subjects changes in adipose gene expression between intervention groups will be evaluated using RNAseq

Measurement of changes to objective sleep will be determined using continuous 24-h EEG monitoring at regular intervals throughout the protocol (including baseline, head down tilt, and recovery).

Changes in subjective sleep quality will be measured using the Karolinska sleepiness scale (KSS) four times per day (mealtimes and bed time) doing every day of the protocol.

Changes to the circadian rhythms of melatonin and cortisol will be measured by radioimmunoassay on saliva samples taken hourly throughout the day at regular intervals throughout the protocol (including baseline, head down tilt, and recovery; same as for EEG).

Changes to the circadian rhythmicity of the whole-blood transcriptome will be measured by whole-genome microarray analyses of RNA extracted from six 4-hourly blood samples taken over a 24-h period at regular intervals throughout the protocol (including baseline, head down tilt, and recovery; same as for EEG).

Vastus Lateralis needle biopsies will be performed before, at the end of the Head Down Tilt period (after about 60 days of bedrest) and of the recovery phases. Change in levels of S-nitrosylated proteins will be measured in active vs. atrophic muscle following extended bed rest.

Vastus Lateralis needle biopsies will be performed before, at the end of the Head Down Tilt period (after about 60 days of bedrest) and of the recovery phases. Change in skeletal muscle gene expression will be analyzed by microarray technology.

Bone resorption marker CTX will be analyzed in the aliquots of total 24h-urine, collected at different timepoints of the study (pre, during and after the bedrest phase).

Energy substrates labeled with stable (non radioactive) isotopes will be used to measure intermediary metabolism and fluxes.

In a healthy standing subject, Galvanic Vestibular Stimulation (GVS) evokes a postural sway response directed towards the anode electrode. The direction of the response is fixed in head coordinates and so it will rotate as the head is turned. To determine if bedrest has affected the accuracy and precision of this sway response, we will measure the direction of the ground reaction force (GRF) response evoked by GVS in standing volunteers under different head orientations. The mean direction (accuracy) along with the trial-to-trial variability (precision) of the GRF vector will be separately determined. Any reduction in accuracy or precision will indicate degraded vestibular control of balance.

24h ECG Holters will be done at different timepoints of the study (pre, during and after the bedrest phase). A conventional Holter analysis will be performed using dedicated commercial software. Moreover, the recordings obtained will be analyzed using several advanced signal processing techniques, which will be focused on different aspects of ventricular repolarization.