Experimental design
Twenty-four healthy individuals (16 men and 8 women; 33.3 ± 9.2 years; 175 ± 9 cm; 74.2 ± 10 kg) consented to and were confined to 60-day head-down bed rest (HDBR) as part of a joint study of the European Space Agency (ESA) and the National Aeronautics and Space Administration (NASA), called the Artificial Gravity Bed Rest—European Space Agency (AGBRESA) study. The study took place at the German Aerospace Centre in Cologne, Germany, between March and December of 2019.
The study was organized in two campaigns, each of which consisted of 15 days for baseline data collection (BDC-15 to BDC-1), 60 days of HDBR (HDT1-HDT59), and 14 days of recovery (R + 0 to R + 13). The study was approved by the ethics committee (2018143) of the North Rhine Medical Association (Ärztekammer Nordrhein) in Düsseldorf, Germany, and was registered in the German Clinical Trials Register (DRKS-ID: DRKS00015677).
Sixteen of the participants received 30 min of AG daily via human centrifugation and the other eight served as a control group (6 men, 2 women). The AG group was further divided into two subgroups: those receiving AG in one continuous 30-min bout (AG1; n = 8; 5 men and 3 women) or in 6 × 5-min bouts with 3-min rest in between bouts (AG2; n = 8; 5 men and 3 women). For the first campaign, participants were pseudo-randomly assigned to groups, whereas for campaign 2, assignment was based on demographic balancing particularly with regards to sex, due to the drop out of three women and subsequent replacement during campaign 2.
Each participant received a complete description of the experimental methods and passed the medical and psychological screening criteria. Medical tests for selection were similar to those used in a previous ESA HDBR study (Rittweger et al. 2015). In addition, the participants passed a centrifuge tolerance test (AG2 protocol) to confirm their eligibility. All participants were made aware of their right to withdraw from the experiment at any time.
Head-down bed rest (HDBR)
Six-degree HDBR was performed in conformity with the International Guidelines for Standardization of bed rest studies in the spaceflight context (https://www.nasa.gov/sites/default/files/atoms/files/bed_rest_studies_complete.pdf). Participants followed a day–night cycle of 06:30 a.m. wake-up, and 22:00 p.m. lights-out. During the HDT period, strict bed rest was followed, and all activities were conducted in the HDT position (hygiene, toilet, reading, etc.). Participants were allowed to change position during HDT, ensuring that one shoulder was in contact with the bed at all times, but not to get up, sit, or stand. During this period, participants were monitored by video and staff surveillance to ensure compliance with the protocol. During the ambulatory and rehabilitation periods, the participants were not authorized to leave the research facility. During their free time, they were allowed leisure activities such as reading, playing games, or computer activities. Daytime sleeping or napping was not permitted. The temperature of the residential area within the facility was controlled across both campaigns at around 22.5 °C (Campaign 1: 22.7 ± 1.6 °C; Campaign 2: 22.5 ± 1.6 °C).
Application of short-arm centrifugation
Transfer to the centrifuge was accomplished with a specific gurney, so that the participants remained at − 6° during transport and were then asked to roll over to the centrifuge nacelle without using their leg muscles. During centrifugation, participants were exposed to 1 g at their estimated centre of mass and were instructed to stay calm, to not move the head, and to keep their leg muscles relaxed. A centrifuge run was as follows: acceleration at 5° s−2 for 32–33 s until the target rotation speed was achieved. Rotation at constant velocity then lasted either 30 min (AG1) or 5 min, with a 3-min rest, repeated six times (AG2). After each run, deceleration was at 5° s−2 until a complete stop. Continuous medical monitoring to ensure participant safety was implemented during all centrifuge runs. The time-of-day for centrifugation was randomly assigned on a day-to-day basis.
Estimation of motor unit number and size
Measurements of both MUNIX and MUSIX were made from the m. abductor digiti minimi (ADM) and m. tibialis anterior (TA) on day 5 before HDBR (BDC-5) and on days 4 (HDT4) and 59 (HDT59) of HDBR (Fig. 1). No measurements were taken during the recovery period. First, the compound muscle action potential (CMAP) was recorded (Viking on Nicolet EDX electromyograph, V22, Natus Neurology, Middleton, Wisconsin, USA), followed by the SIP recordings, at a sampling rate of 50 kHz. A 5–1000-Hz band-pass filter setting was employed to offer a stable baseline and eliminate high-frequency noise. For both muscles, the position of the active recording electrode (15 mm × 20 mm; CareFusion, Middleton, Wisconsin, USA) was adjusted until the highest CMAP amplitude was obtained during BDC-5. This position was marked and noted to minimize errors related to differences in electrode placement between subsequent testing sessions. For the ADM, the active electrode was placed over the motor point of the right hypothenar muscle and the reference electrode at the distal phalanx of the little finger. We used cellophane sheets and a permanent marker to mark the position and other skin landmarks, i.e., palmar creases that could be placed over the hand. The position of the active TA electrode was marked taking the cross-point of the vertical distance from the lateral malleolus and the horizontal distance from the tibial crest, and the reference electrode was placed on the patella. Finally, the ground electrode was placed ~ 2 cm under the stimulation point of the ulnar nerve and ~ 5 cm above the ankle malleoli, for the ADM and TA, respectively. The fingers and toes were strapped to inhibit any dynamic movement. Both the ulnar and peroneal nerves were stimulated with rectangular pulses of 0.2 ms, starting at 10 mA and increasing in 5 mA increments until maximal CMAP was achieved. Following CMAP acquisition, the participant was instructed to produce a number of voluntary isometric contractions (VICs) of the respective muscle. Upon instruction, the participant first produced a contraction with maximal effort, termed 10/10, followed by the minimum contraction necessary to create the desired action, i.e., finger abduction or dorsiflexion, termed 1/10. Participants were then asked to cover the range of forces in between, i.e., 2/10, 3/10, etc., ensuring that each number was covered at least twice, with short rests in between. The force was not measured, though, for each contraction, the surface interference pattern (SIP) of the EMG trace was recorded, with each epoch lasting 300 ms.
Data analysis
The CMAP and SIP signals were used to calculate the signals’ area and power (Nandedkar et al. 2004; Viking on Nicolet EDX electromyograph, Natus Neurology). The ‘‘ideal case motor unit count’’ (ICMUC) is computed using Eq. (5) in “Appendix” (Drey et al. 2013). The relationship between ICMUC and SIP area (\( {\text{ICMUC}} = A \cdot ({\text{Area}}({\text{SIP}}))^{\alpha } \)) is modelled by a power function. The values of \( A \) and \( \alpha \) are derived from a regression analysis fitting the power function. The regression curve characterizes the tested muscle. Finally, MUNIX is calculated by:
$$ {\text{MUNIX}} = A \cdot (20\,{\text{mVms}})^{\alpha } . $$
An SIP area value of 20 mVms is chosen, based on (1) the observation that very slight activity, produced by a few motor units, has an SIP area of around 20 mVms (Nandedkar et al. 2010), and (2) the fact that the assumptions of the model are adequately satisfied for an SIP area of 20 mVms.
MUSIX is obtained by dividing the CMAP amplitude by MUNIX: \( {\text{MUSIX}} = \frac{{{\text{Amplitude}}({\text{CMAP}})}}{\text{MUNIX}}. \)
It should be stressed that MUNIX and MUSIX are indices, and not absolute values, for the number and size of MUs. Any within-session variation in CMAP > 10% was excluded from analysis. For all variables, data from BDC-5 are represented as mean ± SD. To compare differences between groups, data at BDC-5 were normalized per participant to their group mean, and data from HDT4 and HDT59 were calculated as a percent change from BDC-5.
Statistical analysis
Normal distribution of data was confirmed by the Kolmogorov–Smirnov test. A one-way ANOVA was used to assess participant characteristics at baseline for differences in age, sex (chi-squared test), height, and weight between groups. Repeated-measures ANOVA was used to determine the change in CMAP, MUNIX, and MUSIX at HDT4 and HDT59 compared to BDC-5, with TIME as a within-participant factor and GROUP as a between-participant factor. TIME * GROUP interactions were also determined. Post hoc tests used a Bonferroni correction to account for testing of multiple pairs. Significance was defined as p < 0.05.