Tolerability of daily intermittent or continuous short-arm centrifugation during 60-day 6o head down bed rest (AGBRESA study).

Artificial gravity through short-arm centrifugation has potential as a multi-system countermeasure for deconditioning and cranial fluid shifts that may underlie ocular issues in microgravity. However, the optimal short-arm centrifugation protocol that is effective whilst remaining tolerable has yet to be determined. Given that exposure to centrifugation is associated with presyncope and syncope and in addition motion sickness an intermittent protocol has been suggested to be more tolerable. Therefore, we assessed cardiovascular loading and subjective tolerability of daily short arm centrifugation with either an intermittent or a continuous protocol during long-term head-down bed rest as model for microgravity exposure in a mixed sex cohort. During the Artificial Gravity Bed Rest with European Space Agency (AGBRESA) 60 day 6° head down tilt bed rest study we compared the tolerability of daily +1 Gz exposure at the center of mass centrifugation, either performed continuously for 30 minutes, or intermittedly (6 x 5 minutes). Heart rate and blood pressure were assessed daily during centrifugation along with post motion sickness scoring and rate of perceived exertion. During bed rest, 16 subjects (6 women, 10 men), underwent 960 centrifuge runs in total. Ten centrifuge runs had to be terminated prematurely, 8 continuous runs and 2 intermittent runs, mostly due to pre-syncopal symptoms and not motion sickness. All subjects were, however, able to resume centrifuge training on subsequent days. We conclude that both continuous and intermittent short-arm centrifugation protocols providing artificial gravity equivalent to +1 Gz at the center of mass is tolerable in terms of cardiovascular loading and motion sickness during long-term head down tilt bed rest. However, intermittent centrifugation appears marginally better tolerated, albeit differences appear minor.

Introduction

Long term space missions elicit multi-system deconditioning including reduced skeletal muscle strength [1], bone mineral density [2], and central blood volume [3-5]. Moreover, sustained cephalad fluid shifts appear to negatively affect ocular health and brain structure, leading to the so-called space associated neuro-ocular syndrome [6, 7]. Furthermore, returning astronauts may experience reduced aerobic capacity [8], and pre-syncopal symptoms indicative of poorer orthostatic tolerance [9]. In an attempt to counter deconditioning on the International Space Station, integrated resistance and aerobic training is prescribed using a number of dedicated devices [10].

Crewmembers train 6–7 days per week with 6–7 resistance and 4–7 cardiovascular sessions per week [11, 12]. Daily training requires approximately 2.5 hours per crewmember including the training, rest periods, and equipment setup, stowage and cleaning. Despite the substantial investment of time, resource and effort this approach is not entirely effective in mitigating musculoskeletal [13], nor aerobic [5, 14] deconditioning, hence physical rehabilitation is required following return to Earth. Moreover, no effective countermeasures against space associated neuro-ocular syndrome currently exist. Thus more effective and ideally more efficient countermeasures are required for future missions to the Moon, and beyond [15].

Artificial gravity through axial acceleration generated by short-arm human provides musculoskeletal loading via the generation of ground reaction forces and an orthostatic challenge through a hydrostatic pressure gradient both of which are absent in microgravity. Indeed, short-arm human centrifugation may attenuate bone, muscle, and cardiovascular deconditioning [16] induced by 6° head-down bed rest. For instance, in short term (5 day) bed rest, an established terrestrial model of cephalad fluid shifts and space-associated deconditioning [17]) studies, daily 30 minutes centrifugation with at least 1 g at the center of mass resulted in no change in postural muscle strength with good tolerability [18, 19]. Furthermore, exposure to artificial gravity appeared to provide protection against post-bed rest orthostatic intolerance [20, 21]. The primary objective of the AGBRESA bed rest study is to compare the protective effects of one single daily bout (30 min) versus multiple daily bouts of AG (6 x 5 min) on physiological functions that are affected by simulated weightlessness during 60 days of bed rest.

However, exposure to exaggerated hydrostatic pressure gravitational gradients induced by centrifugation can elicit presyncopal symptoms or syncope [20, 22, 23]. Furthermore, head movements within a rotating environment are associated with motion sickness symptoms [24]. Yet, to be acceptable as an integrative spaceflight countermeasure, a form of repeated exposure to artificial gravity needs to be tolerable over a long duration mission for both males and females as a number of recent studies have observed sex differences in autonomic cardiovascular control during exposure to orthostatic stress [22, 25–27].

Thus, the aim of our study was to assess the tolerability of daily 30 minute intermittent, or continuous short-arm centrifugation with 1 g at center of mass during 60 days (6o) head-down bedrest in a mixed sex cohort.

Methods

Study subjects

This study is part of the NASA/ESA/DLR 60-day 6o head down bed rest study ‘Artificial Gravity Bed Rest with European Space Agency’ (AGBRESA) that was conducted from March until December 2019 at the: envihab facility of the Institute of Aerospace Medicine of the German Aerospace Center (DLR) in Cologne, Germany. The study enrolled 24 healthy individuals (16 men, 8 women), who had been submitted to detailed medical and psychological screening having provided written informed consent. The study was approved by the North Rhine Medical Association (2018143 vote from 17.08.2018).

Protocol

Following a 14-day baseline data collection period, study subjects entered 60 days of strict 6° head-down bed rest. At the end of the baseline data collection phase, participants were pseudo-randomly distributed into 3 groups: a control group with no centrifugation, an intermittent centrifugation group, and a continuous centrifugation group. The intermittent centrifugation group underwent daily 6x5 minutes centrifugation with 3 minutes breaks between runs (Fig 1: Left Panel). The continuous centrifugation group underwent a single daily 30 minute centrifugation run (Fig 1: Right Panel).

Fig 1

Artificial gravity was generated by centrifugation with +1Gz at center of mass and approx. +2Gz at feet.

Participants were randomly assigned to an intermittent centrifugation group with 6 x 5 min centrifugation with 3 minute breaks (left side) and a continuous group with 30 min centrifugation (right side).

All centrifugation was performed using the: envihab short-arm human centrifuge with participants exposed to +1 Gz at their center of mass (CoM) and thus approximately +2 Gz at foot level. Rotational speed of the centrifuge was calculated individually based upon each subject’s anthropometry to determine center of mass (ratio center of mass to body height 56% for male/ 54% for female). During ramp up/down phases, (de)acceleration did not exceed 5° s-2 to reduce the risk of vestibular-induced tumbling sensations. All subjects underwent two centrifuge familiarization sessions prior to bed rest at the same +Gz level as the main study with an intermittent profile of two 5 minute periods separated by a 3 minute break.

Head restrainers were not provided, but participants were instructed to keep their body and head still throughout the centrifugation as much as possible. To assist in maintaining consciousness and limit pre-syncopal symptoms, subjects were trained, prior to the bed rest campaign, in the performance of voluntary isometric calf muscle pump contractions along with (the trunk and gluteal muscles) to promote venous return [28]. However, subjects were instructed to contract only when experiencing significant (pre-syncopal) symptoms such as dizziness or blurred vision.

Cardiovascular monitoring

During centrifugation, heart rate was continuously recorded via a five lead electrocardiogram in addition to periodic brachial blood pressure (Philips IntelliVue® MP2). In the intermittent centrifugation group, blood pressure was recorded 2 minutes after each plateau (+1 Gz at center of mass), and in the continuous centrifugation group 2 minutes after the plateau was achieved and every 5 minutes thereafter. Mean heart rate, systolic and diastolic blood pressure were calculated for each first measurement intervals during centrifugation on bed rest days 1, 30 and 60 and compared between intervention groups.

Documentation of all adverse events including premature stops, pre-syncopal signs or cardiac dysrhythmias was performed to facilitate evaluation of tolerability.

Subjective tolerability assessment

General motion sickness susceptibility questionnaire short-form (MSSQ-SF) [29] was determined prior to the head down tilt bed rest including both childhood (MSA) and adulthood (MSB) sub-scores. In both centrifuge groups, Subjective Motion Sickness Ratings (MS: 0 “I am feeling fine” to 20 “I am about to vomit”) [30] and rate of perceived exertion (RPE: 6 “No exertion at all” to 20 “Maximal exertion”) [31] directly after every centrifuge run during bed rest were recorded. Furthermore, Motion Sickness Assessment Questionnaire (MSAQ), Positive and Negative Affect Schedule (PANAS) and Epworth Sleepiness Scale (ESS) were obtained on a weekly basis directly before, and after centrifugation. MSAQ was employed to determine (1 to 9 max) various dimensions (e.g. gastrointestinal) of motion sickness [32]. PANAS was used to assess the effect of centrifugation upon mood. Participants rated each item on a Likert scale from 1 “not at all” to 5 “very much”. The ESS (via rating from 0 (non-) to 3 “high chance of dozing” in 8 contexts) was used to evaluate “drowsiness” since it is a cardinal symptom of motion sickness [33-35]. Furthermore, whenever a centrifuge run was terminated prematurely, the reason was recorded.

Statistical analysis

Generalized linear mixed models with auto-regressive error AR (1) were used to determine if there was an effect of bed rest (time effect) and intervention (intermittent vs. continuous group). Mean values were reported with standard deviation. All residual plots were evaluated using Kolmogorov-Smirnov with none displaying large deviations from normality. All statistical tests were conducted using IBM SPSS version 21 (IBM Corp., USA) with α < 0.05 indicating statistical significance.

Results

The average spin rate required to generate +1 Gz at the center of mass was 30.5 ± 1.0 rpm with radii within 1729–2113 mm at the foot plate. The 16 participants allocated to the two centrifuge groups comprised 10 men and 6 women (71.6 ± 7.4 kg, 33 ± 9.9 yrs, 173 ± 8.8 cm) who experienced 960 centrifuge runs in total.

No serious adverse medical events occurred. However, a total of 10 centrifuge runs (1%, involving 6 different subjects) had to be terminated prematurely; eight runs in the continuous group and two runs in the intermittent group (Fig 2). Of the 10 terminated runs, seven runs–five in the continuous group and two in the intermittent group—had to be terminated due to pre-syncopal signs or symptoms, including significant drop of blood pressure, reporting of tunnel vision and/or lightheadedness. Only one centrifuge run in the continuous group had to be stopped due to severe motion sickness (subsequent MS score of 18/20). Two runs in the continuous group had to be terminated prematurely due to pain resulting from a recent muscle biopsy procedure performed for a different experiment within the bed rest campaign.

Fig 2

Premature terminations of centrifuge sessions.

No clinically significant cardiac dysrhythmias were observed during centrifugation. During continuous centrifugation, two participants demonstrated frequent isolated premature ventricular complexes on 14 non-consecutive days between bed rest days 5 and 51. Two participants in the continuous group and one in the intermittent centrifugation group exhibited occasional premature atrial complexes, but there was no apparent increase in incidence over time. All subjects were, however, able to resume centrifuge training on subsequent days after a termination.

Comparisons of the initial cardiovascular reactions after 2 minutes of centrifugation on bed rest days 1, 30 and 60 revealed significant effects during bed rest for mean heart rate. Mean heart rates were significantly affected by time for the continuous (F = 14.950, p < 0.001, dfs = 14.073) but not for the intermittent group during bed rest (F = 1.558, p = 0.242, dfs = 15.281). Thus mean heart rate was numerically higher in the continuous group on bed rest day 60 but not significant (continuous group: 100.5 ± 18.5 vs. intermittent group: 86.9 ± 5.9, (t (14) = -1.986, p = 0.67) (Table 1). We observed no significant differences in systolic and diastolic blood pressure.

Table 1

Comparison of mean values for heart rate, systolic and diastolic blood pressure during the first 2 minutes of centrifugation at the beginning, middle and end of bed rest.

	Bed rest phase
	Begin		Middle		End
	Intermittent group	Continuous group	Intermittent group	Continuous group	Intermittent group	Continuous group
Heart rate	80.3 ± 8.4	82.4 ± 14.9	86.3 ± 12.7	99.13 ± 18.6	86.9 ± 5.9	100.5 ± 18.5
Systolic blood pressure	119.3 ± 13.4	111.9 ± 41	122.3 ± 10.4	128.8 ± 7.5	130.5 ± 13.5	132.4 ± 11.1
Diastolic blood pressure	76.6 ± 5.9	80.8 ± 9.5	81.8 ± 4.5	84.3 ± 6.3	88.1 ± 8.2	93.8 ± 11.9

Overall MSSQ scores were similar (p = 0.211) prior to bed rest with 3.5 ± 5.4 (MSA 1.9 ± 2.7; MSB 1.6 ± 2.7) for the intermittent centrifugation, 6.0 ± 3.7 (MSA 2.9 ± 2.4; MSB 3.1 ± 2.1) for the continuous centrifugation, and 4.7 ± 4.1 (MSA 2.8 ± 2.8; MSB 1.9 ± 3.0) for the control group.

Daily motion sickness scores were significantly higher in the continuous centrifugation group during bed rest (F = 92.8, p = 0.001, dfs = 202.5) with no effect of bed rest time (F = 0.268, p = 0.605, dfs = 217.2) (Fig 3). Pairwise comparison revealed higher motion sickness scores in the continuous (3.05 ± 0.11) compared to the intermittent centrifugation group (1.58 ± 0.11) (p = 0.001).

Fig 3

Mean values with standard error of daily Motion Sickness (MS) rating immediately following intermittent and continuous centrifugation during 60 day bed rest in the intermittent and in the continuous centrifugation group.

No significant differences in RPE, MSAQ, PANAS or ESS scores were observed during the bed rest phase neither in either nor between groups (Fig 4, Table 2).

Fig 4

Mean values with standard error of daily Rating of Perceived Exertion (RPE) rating immediately following intermittent and continuous centrifugation during 60 day bed rest in the intermittent and in the continuous centrifugation group.

Table 2

Comparison of tolerability assessment (MSAQ, ESS, PANAS) of both centrifuge intervention groups at the beginning, middle and end of bed rest.

	Bed rest phase
	Begin		Middle		End
	Intermittent group	Continuous group	Intermittent group	Continuous group	Intermittent group	Continuous group
MSAQ
ߦOverall	21.96 ± 6.7	20.66 ± 3.7	16.32 ± 2.2	21.76 ± 6.1	19.71 ± 4.4	17.89 ± 2.5
ߦGastrointestinal	21.18 ± 8.9	21.53 ± 4.5	12.50 ± 1.0	22.22 ± 7.6	14.93 ± 3.1	18.06 ± 3.4
ߦCentral	18.06 ± 6.0	16.95 ± 3.3	13.89 ± 4.1	17.52 ± 5.7	16.11 ± 6.0	14.17 ± 2.5
ߦPeripheral	21.30 ± 5.5	25.92 ± 8.2	15.74 ± 0.6	28.39 ± 9.9	20.37 ± 3.7	18.05 ± 1.8
ߦSopite-related	28.12 ± 7.9	20.49 ± 3.9	23.61 ± 4.1	21.61 ± 3.5	28.47 ± 6.4	22.22 ± 3.4
ESS	12.5 ± 1.3	12.25 ± 1.2	15.25 ± 2.3	12.56 ± 1.4	14.25 ± 2.0	13.36 ± 1.8
PANAS (Positive Affect)	23.88 ± 2.6	25.75 ± 2.7	24.5 ± 2.0	24.11 ± 2.0	24.50 ± 3.1	22.88 ± 3.2
PANAS (Negative Affect)	15.50 ± 1.6	13.63 ± 0.5	13.13 ± 0.4	14.44 ± 1.1	15.00 ± 1.2	14.25 ± 0.8

Discussion

We evaluated the tolerability of daily artificial gravity via short-arm centrifugation as a potential countermeasure against deconditioning induced by 60 day bed rest provided either as a single 30 min run or as 6x5 minute runs. Our main findings were that both centrifuge interventions were well tolerated (in both males and females), with no serious adverse events and <1% run termination due to pre-syncopal signs. Only a single run was stopped due to motion sickness, with two terminated due to pain from an experimental procedure from another protocol. All subjects were, however, able to resume centrifuge training on subsequent days. Daily motion sickness scores were low, but significantly higher in the continuous group across bed rest. MSAQ, PANAS or ESS scores were low in both centrifugation groups with no difference between groups indicative of good long-term tolerability.

Short-arm centrifugation induces an orthostatic stress on the cardiovascular system that markedly differs from standing on Earth. While the body experiences 1 g terrestrial gravity throughout with standing, the gravitational stimulus increases in a graded fashion from the head towards the feet during short-arm centrifugation [1].

Yet, previous studies have not observed major differences in cardiovascular regulation when standing and during short-arm centrifugation [36]. In our study, pre-syncope occurred in only a few runs and we did not observe overt syncope. Pre-syncope did occur slightly more frequently in the continuous centrifugation group, suggesting that the breaks in the intermittent protocol may contribute to improved orthostatic tolerance during centrifugation. However, in both groups the incidence was very low, potentially due to the fact that subjects were permitted to perform isometric leg muscle pump exercises when experiencing symptoms. In the absence of countermeasures, bed rest deconditioning is associated with markedly reduced orthostatic tolerance [37]. Interestingly, we did not observe worsening tolerability of short-arm centrifugation over time suggesting that daily artificial gravity may have maintained orthostatic tolerance but this requires further evaluation including specific testing of orthostatic tolerance during bed rest [19].

While we did not observe higher degree cardiac dysrhythmias during centrifugation, frequent isolated premature ventricular complexes in two participants in the continuous centrifugation group are noteworthy as long-arm centrifugation nor orthostatic stress imposed by standing are associated with cardiac dysrhythmias in otherwise healthy persons [38, 39]. Whether premature ventricular complexes were triggered by short-arm centrifugation or other stresses resulting from the complex multi-experimental study cannot be discerned. It is reassuring that orthostatic stress imposed by standing or long-arm centrifugation rarely produces significant cardiac dysrhythmias in otherwise healthy persons [38, 39]. While presyncope occurred slightly more frequently in the continuous centrifugation group the incidence is too small to perform a comprehensive study on intervention group effects. Premature termination of a centrifugation runs were also (albeit rarely) caused by pain due to muscle biopsy from another experiment that were also associated with higher perceived exertion ratings on bed rest days 6 and 55, corroborated by subject comments documented by the attending physician.

As the objective of the present study was to expose subjects to +1 Gz at the center of mass and approximately +2 Gz at the level of the feet, spin rates during centrifugation were relatively high. During such spin rates head movements can exacerbate motion sickness due to induced conflicts between acceleration (gravity) perception and other sensory inputs [24, 40–42]. However, in our study these spin rates were well tolerated even without physical head restraint or head cover to put subjects into darkness which is commonly used. Remarkably, despite the fact that participants were requested, but not physically prevented from moving the head, only a single centrifuge run was stopped due to severe motion sickness symptoms. Indeed, daily ratings for motion sickness did not indicate increases over time in discomfort due to centrifugation-induced cross-coupled sensations.

Thus, this suggests that by limiting centrifugal acceleration to 5° s-2 the risk of significant motion sickness is low, even in the intermittent group whom were exposed to multiple acceleration and decelerations within each session. Thus, why higher (albeit not high) motion sickness ratings were reported in the continuous group is unknown and warrants further study–particularly as MSAQ scoring did not differ significantly between groups. Potential limitations of our study are overestimation of questionnaire results as direct comparison with the control group were not obtained due to the complexity of the study. Although our results may be in accordance with other studies showing high levels of vestibular adaption to high speed short radius rotations over time [12, 30, 43] that may also underlie the low scores for PANAS negative affects–suggesting potentially good long-term tolerability.

In conclusion short-arm centrifugation was well tolerated (in both males and females) during 60-days of 6o head-down tilted bedrest. 30 minute intermittent centrifugation appears to be slightly better tolerated compared to equivalent continuous centrifugation indicated by lower motion sickness scores and fewer run terminations. However, the differences were small and require further study in a mixed sex cohort both as ‘passive’ countermeasures and potentially with concurrent exercise as this may augment effectiveness against multi-systems de-conditioning.

AGBR_quest_results: List of all results from questionnaires ESS, MSAQ and PANAS in a comprehensive manner.

(XLSX)

Click here for additional data file.

Medical data: List of all medical data including heart rate and blood pressure pre and post centrifugation as well as within first two minutes during centrifugation.

(XLSX)

Click here for additional data file.

MS scoring HDT: Recording of motion sickness questionnaires during 60 days of head down tilt bed rest.

(CSV)

Click here for additional data file.

RPE scoring HDT: Recording of perceived exertion questionnaires during 60 days of head down tilt bed rest.

(CSV)

Click here for additional data file.

1 Jul 2020

PONE-D-20-13193

Tolerability and acceptability of daily intermittent or continuous short-arm centrifugation during 60-day 6o head down bed rest

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Reviewer #1: The authors evaluate tolerability and acceptability of intermittent VS continuous short-arm centrifugation during long term head down bed rest. They conclude that both are tolerated, continuous a bit less.

The manuscript is well written but I have a lot of concerns.

First a control group is mentionned but not reported. I would say that before continuous Vs intermittent, the tolerability of daily centrifugation was the main question.

By the way, tolerability /acceptability, can you explain the difference ? Acceptability is not defined in the methods section.

1G was set at the center of mass. Why ? why not HIL, vestibular level, center of bones mass ? how was the center of mass determined ?

Pain is usually evaluated through 10 cm visual analog scale, this a especially important for repeated evaluation to prevent memorisation of the previous score. why did you choose a 0 to 20 scale which could potentially blur longitudinal scoring ?

MSSQ: Your reference is that of Golding that is presumably concerning the MSSQ-short questionnaire, I think you should define MSA and MSB for non-specialists.

At some days it seems that the RPE scoring is higher (see for instance day 55) Can you comment on how other experiments conducted on these subjects (biopsy...) may have influenced tolerability ?

Reviewer #2: PONE-D-20-13193

GENERAL

This report appears to be the first of a number of reports that are based on the AGBRESA study, in which many research groups have studied various aspects of space deconditioning and the effects of artificial gravity as a countermeasure. In order to make these data available in a sound way, too many and too thin “salami” papers should be avoided. This reviewer therefore requests an over-all publication plan (confidential, not to be included in the paper).

The term AGBRESA should be included such that it can be searched (abstract? title?), thereby allowing readers to get the full picture of the AGBRESA results.

SPECIFIC (line numbers introduced by the reviewer- should have been there in the first place)

40: during daily?

104-105 and Fig 1: Is it the area under the curves or the times at top spin rate that are identical between the continuous and the intermittent AG?

110: Radius at the foot plate? (could be computed by the reader from the spin rate, but that should not be necessary)

162-163: Foot pain has been reported in earlier AG studies.

177: p=0.67? tendency?

217: Is ref # 21 really relevant here? Do you mean #19? Missing original rference?

256: Promissory notes regarding future studies are discouraged. Please rephrase.

Reviewer #3: This manuscript addresses an important research question: type of AG to use (that is, intermittent or continuous). I have some suggestions as well as additional references that should be incorporated before this manuscript can be accepted.

1. A lot of text is provided about SANS. You neither measure it nor discuss your results in the context of SANS. I would suggest that you expand the introduction along the lines of the responses that you measured (hemodynamic, vestibular, etc.). Additional references should be placed in. For instance, Iwase et al., 2002; Goswami et al., 2015a; White et al., 2015)

2. Throughout the paper, no mention is made about gender. Then suddenly in the study results, we observe that the study involved males and females. The authors should discuss in the introduction what is the current evidence about sex-based differences in responses in space and/or during AG exposure. See, for example, Evans et al., 2018. Then in the discussion, the results obtained here (sex differences in the responses) should be thoroughly discussed.

3. If you were asked by a researcher, which of these protocols to use for future AG studies, what would you recommend? This could be a clear message of the paper and should be discussed at length in the paper. Currently, it is not clear which AG protocol should be used in the future.

4. Could you also speculate which of these AG protocol would you recommend in the future if additional countermeasures such as nutrition or exercise are also added? See Iwase et al., 2005.

5. Why did some of your participants faint/ develop presyncopal symptoms during AG exposure? Have you tested what their known limit of AG tolerance was? Recent evidence suggests that people take different times to develop presyncope on the centrifuge (Goswami et al.2015b). You should discuss your results in context to this paper as well as come up with clear recommendations about which AG protocol to use based on your evidence as well as published work.

I congratulate the authors for their creativity and thank them for advancing the field of AG.

References in detail

Evans, JM et al. Artificial Gravity as a Countermeasure to the Cardiovascular Deconditioning of Spaceflight: Gender Perspectives. Front Physiol. 2018; 9: 716-716.

Evans, JM et al. Hypovolemic men and women regulate blood pressure differently following exposure to artificial gravity. Eur J Appl Physiol. 2015; 115(12):2631-2640

Goswami et al. Short-arm human centrifugation with 0.4g at eye and 0.75g at heart level provides similar cerebrovascular and cardiovascular responses to standing. Eur J Appl Physiol. 2015a; 115(7):1569-1575

Goswami et al. Effects of individualized centrifugation training on orthostatic tolerance in men and women. PLoS One. 2015b; 10(5):e0125780-e012578

Iwase et al. Effects of Graded Load of Artificial Gravity on Cardiovascular Functions in Humans. Environ Med 2002 Dec;46(1-2):29-32.

White et al.The effects of varying gravito-inertial stressors on grip strength and hemodynamic responses in men and women. Eur J Appl Physiol. 2019; 119(4):951-960

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Reviewer #3: No

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27 Jul 2020

Response to Reviewer

Dear Reviewer,

Dear Editor,

Thank you for your helpful comments on our manuscript. We have read them carefully and worked on a revised version that hopefully complies to your requirements. Please find a list of changes based on your input and comments below:

Editor comments:

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly.

Answer:

1.-2.: We have changed all declared parts including funding statement, author’s contribution and competing interests as requested.

3. We have added captions and a short description of all Supporting Information files.

Reviewer#1 comments:

1. The authors evaluate tolerability and acceptability of intermittent VS continuous short-arm centrifugation during long term head down bed rest. They conclude that both are tolerated, continuous a bit less. The manuscript is well written but I have a lot of concerns. First a control group is mentioned but not reported. I would say that before continuous Vs intermittent, the tolerability of daily centrifugation was the main question.

Answer: Thank you for pointing this out. Since daily centrifugation has not previously been tested in a longer-term bedrest study, we were concerned that adverse effects, such as syncope, could limit the utility of the intervention with increasing deconditioning and volume changes. If so, the countermeasure would be difficult to conduct during longer term space missions. Thus, from a practical point of view, the main question was how many centrifugation sessions would have to be aborted. Because the control group did not undergo centrifugation, there cannot be an aborted centrifugation. However, some of the more subjective tolerability assessments could be overestimated without adjustment of these measures in the control group. Unfortunately, these measurements were not obtained in the control group due to complexity of the study. We included the issue as potential limitation in the discussion (see lines 261-262).

2. By the way, tolerability /acceptability, can you explain the difference? Acceptability is not defined in the methods section.

Answer: We have changed the terminus to tolerability as this is much clearer.

3. 1G was set at the center of mass. Why? why not HIL, vestibular level, center of bones mass ? How was the center of mass determined ?

Answer: The centrifuge profile was a priori decided by NASA and ESA together with the DLR centrifuge science team. Previous centrifuge studies such as the BRAG1 study at MEDES (see Clement et al. 2015) showed good tolerability and partially effectiveness of both protocols during 5 days head down tilt bed rest. We have added a brief explanation in line 198-200. The setting in MEDES for BRAG1 was CoM at approximately 1.18 – 1.22 m from the CoR (Center of Rotation). The resulting rotational speed was 27.8 – 28.2, resulting in a +1Gz at CoM and approx. +2Gz at the feet. During AGBRESA we determined the center of mass of the subjects using anthropometry. Rotational speed of the centrifuge was therefore calculated for each subject individually. We also calculated and changed the position of each subject along the centrifuge radius to meet +2Gz at feet accordingly (see lines 112-120).

4. Pain is usually evaluated through 10 cm visual analog scale, this a especially important for repeated evaluation to prevent memorisation of the previous score. why did you choose a 0 to 20 scale which could potentially blur longitudinal scoring ?

Answer: We recorded motion sickness using a 0 to 20 scale (0 “I am feeling fine” to 20 “I am about to vomit”) that was fast and easy to use during centrifugation according Young et al. 2003. Pain was recorded only by the attending physician when reported by the subject and was not part of our experiment. As reported pain from other experiments (e.g. previous muscle biopsy) caused premature stops of intervention.

5. MSSQ: Your reference is that of Golding that is presumably concerning the MSSQ-short questionnaire, I think you should define MSA and MSB for non-specialists.

Answer: Thank you. We have added a short definition of MSA and MSB in the method section (see lines 138-140)

6. At some days it seems that the RPE scoring is higher (see for instance day 55) Can you comment on how other experiments conducted on these subjects (biopsy...) may have influenced tolerability ?

Answer: We have checked RPE scoring and the documentation of participant’s comments on pain during centrifugation after muscle biopsy experiments and have added this point to the discussion (lines 241-244).

Reviewer#2 comments:

7. This report appears to be the first of a number of reports that are based on the AGBRESA study, in which many research groups have studied various aspects of space deconditioning and the effects of artificial gravity as a countermeasure. In order to make these data available in a sound way, too many and too thin “salami” papers should be avoided. This reviewer therefore requests an over-all publication plan (confidential, not to be included in the paper).

Answer: ESA and NASA selected principal investigators for the AGBRESA study using a peer review approach. Each principal investigator has the right to publish experiments conducted as part of the AGBRESA study, however, there are some differences between NASA and ESA regulations. There is no overall-publication plan.

8. The term AGBRESA should be included such that it can be searched (abstract? title?), thereby allowing readers to get the full picture of the AGBRESA results.

Answer: In accordance with the comment above we have included the term AGBRESA also in the title.

9. SPECIFIC (line numbers introduced by the reviewer- should have been there in the first place)

40: during daily?

Answer: Corrected, thank you.

104-105 and Fig 1: Is it the area under the curves or the times at top spin rate that are identical between the continuous and the intermittent AG?

Answer: Due to the study design the times at top spin rate were identical, so both groups were exposed to daily 30 minutes AG with +1Gz at CoM. The intermittent group had a longer exposure to AG due to the frequent ramp up/down.

110: Radius at the foot plate? (could be computed by the reader from the spin rate, but that should not be necessary)

Answer: We calculated an individual centrifuge speed and position along centrifuge radius to ensure +1Gz at CoM and +2Gz at the foot plate. These radii were within 1729-2113 mm (see lines 112-120). E. Mulder et al. will publish a more general method paper elsewhere to give further details.

162-163: Foot pain has been reported in earlier AG studies.

177: p=0.67? tendency?

Answer: We changed the sentences to be more precise.

217: Is ref # 21 really relevant here? Do you mean #19? Missing original reference?

Answer: You are right, we changed the relevant reference.

256: Promissory notes regarding future studies are discouraged. Please rephrase.

Answer: As suggested, we rewrote the section.

Reviewer #3 comments:

1. A lot of text is provided about SANS. You neither measure it nor discuss your results in the context of SANS. I would suggest that you expand the introduction along the lines of the responses that you measured (hemodynamic, vestibular, etc.). Additional references should be placed in. For instance, Iwase et al., 2002; Goswami et al., 2015a; White et al., 2015)

Answer: Thank you. We have added missing sentences and references in the introduction.

2. Throughout the paper, no mention is made about gender. Then suddenly in the study results, we observe that the study involved males and females. The authors should discuss in the introduction what is the current evidence about sex-based differences in responses in space and/or during AG exposure. See, for example, Evans et al., 2018. Then in the discussion, the results obtained here (sex differences in the responses) should be thoroughly discussed.

Answer: We reported gender distribution of the study in the method section (see line 101) and have added recent findings from Evans et al. 2018 in the introduction. We discussed internally a sex based analysis of our results but the limited sample size (n = 8 per group) do not allow well powered statistics about gender differences. This topic was therefore also discussed with ESA. For the two upcoming bed rest studies involving AG as intervention will use two groups with n = 12 participants each. We also concluded in the discussion that a mixed sex cohort is required to further investigate AG as potential countermeasure.

3. If you were asked by a researcher, which of these protocols to use for future AG studies, what would you recommend? This could be a clear message of the paper and should be discussed at length in the paper. Currently, it is not clear which AG protocol should be used in the future.

Answer: You are right. We have rewritten and added additional text in the discussion section.

4. Could you also speculate which of these AG protocol would you recommend in the future if additional countermeasures such as nutrition or exercise are also added? See Iwase et al., 2005.

Answer: We have rewritten the discussion to make the planned combination of AG+exercises more clear.

5. Why did some of your participants faint/ develop presyncopal symptoms during AG exposure? Have you tested what their known limit of AG tolerance was? Recent evidence suggests that people take different times to develop presyncope on the centrifuge (Goswami et al.2015b). You should discuss your results in context to this paper as well as come up with clear recommendations about which AG protocol to use based on your evidence as well as published work.

Answer: During the Baseline data collection phase of AGBRESA only two short familiarization sessions with centrifugation were allowed for each subject. Therefore individual AG tolerance testing as shown by Goswami et al. was unfortunately not allowed by the study setting. To the authors knowledge this topic is currently under discussion by the ESA bed rest expert team for both upcoming bed rest studies in MEDES and Planica.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

19 Aug 2020

PONE-D-20-13193R1

Tolerability of daily intermittent or continuous short-arm centrifugation during 60-day 6o head down bed rest (AGBRESA study)

PLOS ONE

Dear Dr. Frett,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #3: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: PONE-D-20-13193R1

GENERAL

My comments and questions have been adequately addressed. With respect to a publication plan I note that a method paper by Mulder is planned. Please consider merging that with the present ms for a more solid paper.

SPECIFIC

Line 153: “Statistical analysis. Generalized linear mixed models with auto-regressive error

AR(1) were used to determine if there was an effect of bed rest (time effect) and intervention (intermittent vs. continuous group). Mean values were reported with standard

Deviation”.

This reviewer is not sure that the above statistical approach is appropriate for rated estimates of motion sickness. Please consult an expert. Sorry for not pointing this out in my previous review.

Reviewer #3: Most of my comments have been addressed.

However, reference numbers 21 and 23 are the same.

Reference number 23 should be replaced by: Goswami, N; Blaber, AP; Hinghofer-Szalkay, H; Convertino, VA. Lower Body Negative Pressure: Physiological Effects, Applications, and Implementation. Physiol Rev. 2019; 99(1): 807-851

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Reviewer #2: No

Reviewer #3: No

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24 Aug 2020

Response to Reviewer

Dear Ladies and Gentlemen,

We thank the editor and both reviewers for helpful comments on our manuscript. We reviewed the comments carefully and revised the manuscript accordingly. Please find a list of changes based on your input and comments below:

Reviewer#2 comments:

1. GENERAL

My comments and questions have been adequately addressed. With respect to a publication plan I note that a method paper by Mulder is planned. Please consider merging that with the present ms for a more solid paper.

Answer: We have carefully discussed your suggestion. A method paper about AGBRESA is in preparation. However, the manuscript is rather dense and contains very detailed information regarding study design, including recruitment and methods for standardization, demographics, vital signs, and dietary composition among others. All other data including the bed rest core data will be embedded in manuscripts by principial investigators of individual experiments funded by DLR, ESA or NASA. Given the potential application of artificial gravity as countermeasure in space and a series of future studies testing this modality in combination with exercise, we believe that a detailed analysis of tolerability should be made available to the scientific community.

2. SPECIFIC

Line 153: “Statistical analysis. Generalized linear mixed models with auto-regressive error AR(1) were used to determine if there was an effect of bed rest (time effect) and intervention (intermittent vs. continuous group). Mean values were reported with standard Deviation”. This reviewer is not sure that the above statistical approach is appropriate for rated estimates of motion sickness. Please consult an expert. Sorry for not pointing this out in my previous review.

Answer: Thank you for pointing this out. We have consulted an experienced biometrician prior to the data analysis and have discussed the correct method to analyze the data. The biometrician advised us to use this linear mixed model.

Reviewer #3 comments:

1. Most of my comments have been addressed. However, reference numbers 21 and 23 are the same. Reference number 23 should be replaced by: Goswami, N; Blaber, AP; Hinghofer-Szalkay, H; Convertino, VA. Lower Body Negative Pressure: Physiological Effects, Applications, and Implementation. Physiol Rev. 2019; 99(1): 807-851

Answer: Thank you for this note, we have corrected the error and included the additional reference.

2 Sep 2020

Tolerability of daily intermittent or continuous short-arm centrifugation during 60-day 6o head down bed rest (AGBRESA study)

PONE-D-20-13193R2

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Reviewers' comments:

10 Sep 2020

PONE-D-20-13193R2

Tolerability of daily intermittent or continuous short-arm centrifugation during 60-day 6o head down bed rest (AGBRESA study)

Dear Dr. Frett:

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