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DI-5-Cuffs: Bone Remodelling and
Associated Metabolism Markers in
Humans After Five Days of Dry
Immersion to Simulate Microgravity
Marie-Thérèse Linossier
1
*, Laura Peurière
1
, Peter Fernandez
1
, Myriam Normand
1
,
Arnaud Beck
2
, Marie-Pierre Bareille
2
, Christine Bonneau
3
, Guillemette Gauquelin-Koch
4
and Laurence Vico
1
1
INSERM, U 1059, University of Saint-Etienne, University of Lyon, Saint Etienne, France,
2
Institute of Space Physiology and
Medicine (MEDES), Toulouse, France,
3
Biochemical Analysis Laboratory, University Hospital, Saint Etienne, France,
4
French
Space Agency (CNES), Paris, France
Background: The dry immersion (DI) model closely reproduces factors of spaceflight
environment such as supportlessness, mechanical and axial unloading, physical inactivity,
and induces early increased bone resorption activity and metabolic responses as well as
fluid centralization. The main goal of this experiment was to assess the efficacity of
venoconstrictive thigh cuffs, as countermeasure to limit cephalad fluidshift, on DI-induced
deconditioning, in particular for body fluids and related ophthalmological disorders. Our
specific goal was to deepen our knowledge on the DI effects on the musculoskeletal events
and to test whether intermittent counteracting fluid transfer would affect DI-induced bone
modifications.
Methods: Eighteen males divided into Control (DI) or Cuffs (DI-TC) group underwent an
unloading condition for 5 days. DI-TC group wore thigh cuffs 8–10 h/day during DI period.
Key markers of bone turnover, phospho-calcic metabolism and associated metabolic
factors were measured.
Results: In the DI group, bone resorption increased as shown by higher level in Tartrate-
resistant acid phosphatase isoform 5b at DI
24h
. C-terminal telopeptide levels were
unchanged. Bone formation and mineralization were also affected at DI
24h
with a
decreased in collagen type I synthesis and an increased bone-specific alkaline
phosphatase. In addition, osteocalcin and periostin levels decreased at DI
120h
.
Calcemia increased up to a peak at DI
48h
, inducing a trend to decrease in parathyroid
hormone levels at DI
120h
. Phosphatemia remained unchanged. Insulin-like growth factor 1
and visfatin were very sensitive to DI conditions as evidenced by higher levels by 120% vs.
baseline for visfatin at DI
48h
. Lipocalin-2, a potential regulator of bone homeostasis, and
irisin were unchanged. The changes in bone turnover markers were similar in the two
groups. Only periostin and visfatin changes were, at least partially, prevented by thigh
cuffs.
Conclusion: This study confirmed the rapid dissociation between bone formation and
resorption under DI conditions. It revealed an adaptation peak at DI
48h
, then the
Edited by:
Daniel Bouvard,
UMR5237 Centre de Recherche en
Biologie cellulaire de Montpellier
(CRBM), France
Reviewed by:
Marc Wein,
Massachusetts General Hospital and
Harvard Medical School, United States
Nastassia Navasiolava,
Centre Hospitalier Universitaire
d’Angers, France
*Correspondence:
Marie-Thérèse Linossier
linossier@univ-st-etienne.fr
Specialty section:
This article was submitted to
Skeletal Physiology,
a section of the journal
Frontiers in Physiology
Received: 25 October 2021
Accepted: 07 April 2022
Published: 27 April 2022
Citation:
Linossier M-T, Peurière L,
Fernandez P, Normand M, Beck A,
Bareille M-P, Bonneau C,
Gauquelin-Koch G and Vico L (2022)
DI-5-Cuffs: Bone Remodelling and
Associated Metabolism Markers in
Humans After Five Days of Dry
Immersion to Simulate Microgravity.
Front. Physiol. 13:801448.
doi: 10.3389/fphys.2022.801448
Frontiers in Physiology | www.frontiersin.org April 2022 | Volume 13 | Article 8014481
ORIGINAL RESEARCH
published: 27 April 2022
doi: 10.3389/fphys.2022.801448
maintenance of this new metabolic state during all DI. Notably, collagen synthesis and
mineralisation markers evolved asynchronously. Thigh cuffs did not prevent significantly
the DI-induced deleterious effects on bone cellular activities and/or energy metabolism.
Keywords: simulated microgravity, dry immersion, thigh cuffs, bone remodelling, energy metabolism
INTRODUCTION
Lack of Earth gravity is associated with body deconditioning as
demonstrated by changes in various physiological systems such as
cardiac, muscle and bone functions (Shelhamer, et al., 2020). The
dry immersion (DI) model is a unique analogue of reduced
gravity by providing mechanical and axial unloading,
advanced physical inactivity, as well as a lack of a supporting
structure for the body. Indeed, for subject freely suspended in
water, support load is spread nearly uniformly around the entire
bodysurface. The absence of specific zones that carry the weight
(like the back and sides of the torso in bedrest) creates a support
deprivation state akin to weightlessness. Our previous experiment
had been carried out on 12 subjects placed in immersion
conditions for 3 days. This study allowed us to show that the
effects of DI on musculoskeletal system could be more severe and/
or earlier than in support-loaded bed rest (Demangel et al., 2017;
Linossier et al., 2017). Thus, serum marker of bone resorption
activity as assessed by tartrate-resistant acid phosphatase isoform
5b and N-terminal crosslinked telopeptide of type I collagen
levels increased as early as DI
24h
. At the same time, total
procollagen type I N- and C-terminal propeptides and
osteoprotegerin, representing bone formation markers,
decreased. At the bone level, loss induced by prolonged
microgravity exposure is mainly seen in the weight-bearing
segments of the skeleton, i.e., the lower limbs, with few
differences at the non weight-bearing radius site (Vico et al.,
2017). This differential bone response could be related not only to
greater unloading for weight-bearing sites, but also to
redistribution of tissue fluids towards the thoraco-cephalad
regions. To prevent shift in fluids towards the head, thigh
cuffs are used to sequester blood and fluids in the lower limbs
by compressing blood vessels in the proximal parts of the thighs.
First tested in 1984 during a 232-days space flight, thigh cuffs
have proved effective in alleviating the symptoms associated with
cephalad fluid shift in the early hours and days in space (Arbeille
et al., 1999;Fomina et al., 2004). Therefore, since about thirty
years, thigh cuffs are used by cosmonauts in flight as a routine
countermeasure limiting fluid movement and improving their
comfort. Cardiovascular changes occur early and lead to a new
stable hemodynamic balance after a few days (Buckey, 2006).
Influences of thigh cuffs on the cardiovascular system and
haemodynamic changes have already been well studied during
Head-Down Bed Rest (HDBR) (Arbeille et al., 1999;Custaud
et al., 2000;Millet, et al., 2000;Pavy-Le Traon et al., 2001;Yao
et al., 2008). However, in these studies, their effects are not always
consistent. Based on the first four studies cited, using a thigh
pressure of 30 mm Hg during 10 h daily, the effects of thigh cuffs
on plasma volume, heart rate and spontaneous baroreflex slope
(SBRs) have been proven insufficient to prevent orthostatic
intolerance induced by 7 days of HDBR. Yao et al. (2008)
indicated that daily use of thigh cuffs at 40 mmg Hg (10 h/
day) during 10 days of HDBR did not completely prevent the
decrease in haemodynamics of the right middle cerebral artery,
but, contrary to the other studies, was effective in preventing
orthostatic intolerance. DI induces prompt body fluids
centralization, mainly due to hydrostatic squeezing of
superficial tissues and vessels, and is therefore particularly
adapted for rapid evaluation of countermeasures against fluid
transfer and its consequences. Such countermeasures become a
priority in preparation of deep space missions, since cephalad
fluid shift might contribute to spaceflight associated neuro-ocular
syndrome. This integrative study was primarily designed to test
the efficacy of venoconstrictive thigh cuffs, as countermeasure to
limit cephalad fluidshift, on DI-induced deconditioning, in
particular for body fluids and related ophthalmological
disorders (for results cf. Robin et al., 2020;Kermorgant et al.,
2021). However, several physiological systems interact, including
the cardiovascular and musculoskeletal systems. By revealing that
the skeleton exerts an endocrine regulation of sugar homeostasis,
the Karsenty’s team expands the biological importance of this
organ and the understanding of energy metabolism (Lee et al.,
2007;Schwetz, Verena et al., 2012). The interaction between the
different systems are not only affected by fluid movements and/or
hemodynamic changes, but also by bone factors such as
osteocalcin known for its endocrine role on the energy
metabolism. Therefore, all modifications of fluid transfers
(whether it is a redistribution under microgravity model and/
or a limitation of this redistribution with the addition of Thigh
cuff to DI) can potentially change the effects exerted by the bone
on the metabolism. To date, no study has considered the impact
of thigh cuffs on the bone remodelling and associated metabolism
markers in humans either in real or simulated microgravity. In
rats, it has been shown that femoral vein ligation by increasing
intraosseous pressure, induces increased bone mass in the
hindlimb of suspended animals (Bergula et al., 1999).
Therefore, this 5-day DI offers a unique opportunity to test
thigh cuff effects on the musculoskeletal events. The first aim of this
study was to see if the DI-induced changes persist over a 5 days dry
immersion with the same intensity than the one seen after 3 days.
The daily use of venoconstrictive thigh cuffs should allow us to see
if the limitation of fluid transfer for 10 h/day can modulate the
effects induced by DI on bone remodelling in humans.
MATERIAL AND METHODS
Subjects
Twenty healthy men were recruited. Two subjects withdrew from
the study before the start of their experimental period for reasons
Frontiers in Physiology | www.frontiersin.org April 2022 | Volume 13 | Article 8014482
Linossier et al. DI-5-Cuffs and Bone Remodeling Markers
unrelated to the protocol. A total of eighteen subjects were
included in the study. A period of 4 days before immersion
was applied in order to proceed to the basal data collection
(BDC). Subjects were randomly allotted at BDC-2 to Dry
Immersion (DI) or Dry Immersion with thigh Cuffs (DI-TC)
group (9/9 split). All subjects were informed about the
experimental procedures and gave their written consent. The
experimental protocol conformed to the standards set by the
Declaration of Helsinki and was approved by the local Ethic
Committee (CPP Est III: October 2, 2018, n°ID RCB 2018-
A01470-55) and French Health Authorities (ANSM: August 13,
2018). ClinicalTrials.gov Identifier: NCT03915457.
Baseline group characteristics are detailed in Table 1. There
was no significant difference between groups at baseline.
General Protocol, dry Immersion
Organization, Thigh Cuffs Countermeasure
The study was conducted at the MEDES space clinic, Toulouse,
France from November, 19, 2018 to March, 23, 2019 in a period
lasting 12 days as described in Figure 1. Subjects arrived in the
evening of BDC-5 and left after 48-h of recovery (at R + 2). The
experimental protocol included 4 days of ambulatory baseline
measurements before immersion (BDC-4 to BDC-1), 5 days
(120 h) of dry immersion (DI1 to DI5) and 3 days of
ambulatory recovery (R0, R + 1, R + 2).
A week prior to beginning of the protocol the subjects went to
MEDES for pre-immersion muscle biopsy and resting metabolic
rate measurement.
Subjects of the Cuff group wore the thigh cuffs during the
5 days of DI, from 10:00 to 18:00 at DI1 and from 08:00 to 18:00 at
DI2-DI5. Thigh cuffs were elastic strips, adapted to each subject
to have the same effects on lower-limb distensibility as at
counterpressure of about 30 mmHg. Individual adjustment was
determined for each subject with calf plethysmography,
performed in the supine position at BDC-2. At DI1, thigh
cuffs were put on immediately prior to the onset of immersion
at 10 h. 30 mmHg was selected, as it matches to the initial thigh
cuff pressure used by cosmonauts and the pressure tested and
evaluated during a 7-day HDBR held at MEDES in 1997–1998
(Arbeille et al., 1999;Custaud et al., 2000;Millet, et al., 2000;
Pavy-Le Traon et al., 2001).
A strict DI protocol was conducted according to methodology
detailed previously (De Abreu et al., 2017;Linossier et al., 2017).
Two subjects, one Dry Immersion and one Dry Immersion with
Thigh Cuffs, underwent dry immersion simultaneously in the
same room, in two separate baths (except for two subjects, one DI
and one DI-TC, who had no mate). Thermoneutral water
temperature was continuously maintained at 33 ± 0.5°C. Light-
off period was set at 23:00–07:00. Daily hygiene, weighing and
some specific measurements required extraction from the bath.
During these out-of-bath periods, subjects maintained the -6°
head-down position in order to avoid orthostatic stimulation and
preserve DI effects as much as possible. Total out-of-bath supine
time for the 120 h of immersion was 9.7 ± 1.3 h. On DI1-DI4 out-
of-bath time was 1.1 ± 0.6 h/day. On DI5 out-of-bath time was
5.3 ± 1.1 h, because of muscle biopsy and MRI. Otherwise, during
DI, subjects remained immersed in a supine position for all
activities and were continuously observed by video
monitoring. Body weight, blood pressure, heart rate and
tympanic body temperature were measured daily. Water intake
throughout the protocol was ad libitum between 35–60 ml/kg/day
and was measured. The meals of each experiment day were
identical for all participants and dietary intake was
individually tailored and controlled during the study. VO
2max
bicycle ergometer test was performed in the evening of BDC-2
and R0.
Blood Sampling
Fasting blood samples were collected always at the same time
(around 7.30 am) during the course of the study. As mentioned in
Figure 1, they were taken at baseline 1 day (BDC
-24h
) and just
TABLE 1 | Baseline group characteristics at BDC-2.
Age (y) Height (cm) Weight (kg) BMI (kg/m
2
) (ml/min/kg)
Dry Immersion (DI) n= 9 33.4 ± 7.0 176 ± 6 74.5 ± 7.2 24.2 ± 1.8 46.5 ± 8.1
Dry Immersion with Thigh cuff (DI-TC) n= 9 33.8 ± 4.0 180 ± 4 74.4 ± 9.2 22.8 ± 1.8 46.9 ± 5.8
Values are mean ± SD. Unpaired t-test did not reveal significant difference between groups.
FIGURE 1 | Description of the study. A Time line of the experiment with the timing of different blood samples. Blue area corresponds to the DI phase. BDC: baseline
data collection; DI: dry immersion; R: recovery.
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Linossier et al. DI-5-Cuffs and Bone Remodeling Markers
before the commencement of immersion (BDC
-2h
), after 24 h-
(DI
24h
), 48 h- (DI
48h
), and 120 h- (DI
120h
) immersion, then 48 h
after the return to loading conditions (R
+48h
). Serum was
separated after blood collection on BD Vacutainer
®
SST™
tubes (with clotting activator). BD Vacutainer
®
EDTA K3
tubes were used for plasma generation. Serum and plasma
were frozen at −80°C right away after the centrifugation until
analysis. Measurements were carried out simultaneously in all
samples at the end of the study.
Measurements of Biological Markers
All parameters were measured at BDC
-24h
and BDC
-2h
(2 h before
immersion), then at DI
24h
,DI
48h
,DI
120h
and R
+48h
as indicated in
Figure 1. It should be noted that only the data obtained at BDC
-
24h
were retained for the determination of the baseline level
(BDC) ; indeed, the day before immersion, after the fasting
blood sample taken at BDC
-24h
, a test to measure the
metabolic flexibility by indirect calorimetry during the fasted
to fed transition was made ; this test could potentially be at the
origin of the systematically and significantly lower values at BDC
-
2h
vs. BDC
-24h
recorded for the majority of markers.
Serum samples: C-terminal crosslinked telopeptide of type I
collagen [CTx], procollagen type I N-terminal propeptide
[P1NP], bone alkaline phosphatase [bAP], intact and N-mid
osteocalcin fragment [OC] and insulin-like growth factor 1
[IGF1] were determined by automated chemiluminescence
immunoassay (IDS-iSYS automated analyzer, Boldon,
United Kingdom). The following parameters were measured
by enzyme-immunoassay (EIA) kits: tartrate-resistant acid
phosphatase isoform 5b [TRAP5b] (Microvue Bone, Quidel
Corporation, San Diego, CA, United States );
undercarboxylated [Glu-OC] and carboxylated [Gla-OC] OC
(Takara Bio, Inc., Otsu, Japan); secretory form of nicotinamide
phosphoribosyl-transferase [visfatin] (Adipogen AG, Liestal,
Switzerland); Irisin (Adipogen AG, Liestal, Switzerland);
periostin (Biomedica Medizinprodukte GmbH, Wien, Austria).
Serum intact parathyroid hormone [1–84 PTH], calcium and
phosphorus were measured using electrochemiluminescence
immunoassay (Cobas
®
8000 modular analyzer, Roche
Diagnostics Ltd., Rotkreuz, Switzerland).
Plasma samples: Neutrophil gelatinase-associated lipocalin
[lipocalin-2] was measured also by EIA kit (Epitope
Diagnostics, Inc., San Diego, CA, United States.
Statistical Analysis
In each group, for each parameter, baseline level (BDC) was
defined as the value of the measured variables at BDC
-24h
. Because
of non-normal distributions and small number of subjects, non-
parametric statistics have been performed. Data were expressed as
median and interquartile range (IQR).
In each group, the time effect was assessed using non-
parametric Friedman rank-sum test. Wilcoxon tests
corrected by the fdr adjustment method of Benjamini and
Hochberg (1995) were used for post hoc comparisons between
each timing.
The TC treatment effect was further analyzed at each time
point by Mann-Whitney tests between the 2 groups.
The changes from baseline expressed as percent difference
were plotted at DI
24h
to DI
120h
or R
+48h
for all parameters in
figures for better visualization.
The relationships between changes (expressed vs. BDC
-24h
)in
parameters at DI
48h
or at DI
120h
were investigated using
nonparametric Spearman correlations. All statistical tests were
carried out with the R statistic software supported by the R
Foundation for Statistical Computing. pvalues less than 0.05 were
considered to be statistically significant.
RESULTS
Body Weight and VO
2max
Similar changes were recorded for the two groups. Body weight
was found significantly decreased after 1-day immersion and
remained so during all the immersion period (Table 2). Body
weight did not fully normalize after 2 days reloading.VO
2max
decreased during dry immersion phase by near 10% vs. BDC
values.
Bone Metabolism
For the different markers, data measured at baseline, during the
immersion period and after 24 h of recovery are summarized in
Tables 3 and 4. For all parameters, there was no significant
difference between groups at baseline.
Phosphocalcic metabolism: Calcium significantly increased
as soon as the first day of immersion and reached a peak at DI
48h
in the two groups; at DI
48h
, these levels were higher by 5 and 3%
for DI and DI-TC, respectively, when compared to BDC, this
increase being less in the DI-TC than DI (Figure 2). These levels
remained elevated during all dry immersion phase for DI group
whereas, for DI-TC group, concentrations returned to BDC level
at DI
120h
; at this latter timing, calcium concentrations became
significantly higher for DI vs. DI-TC.
Serum intact PTH levels tend to decrease at DI
120h
by 18% in
DI group (p= 0.064) when compared to BDC while no significant
change was observed in DI-TC group. Phosphatemia was
unchanged during the immersion phase in the two groups.
Bone resorption activity: Serum TRAP5b significantly
increased as soon as the first day of immersion and remained
elevated during the entire dry immersion phase (by 15 and 13% at
DI
48h
for DI and DI-TC, respectively when compared to BDC) in
both groups (Figure 3). After the DI period, at R
+48h,
TRAP5b
remained high in the DI-group while in the DI-TC it began to
decrease, the difference between the two groups being not
statistically different. No change in serum CTx was seen in the
two groups (Table 3).
Bone formation activity: P1NP decreased progressively
during all immersion periods in the two groups; at DI
48h
, the
decrease appeared slightly smaller for DI vs. DI-TC although not
significantly (p= 0.063); at DI
120h
, levels were significantly lower
by 15 and 21% for DI and DI-TC, respectively when compared to
BDC (Figure 3). Serum bAP concentrations increased as early as
1-day immersion and reached a peak at DI
48h
; this increase
appeared slightly higher for DI when compared to DI-TC
although not significantly (15 vs. 8%, respectively, p= 0.063) ;
Frontiers in Physiology | www.frontiersin.org April 2022 | Volume 13 | Article 8014484
Linossier et al. DI-5-Cuffs and Bone Remodeling Markers
levels remained elevated during all dry immersion phase for DI
group wheras, for DI-TC group, concentrations returned to BDC
level at DI
120h
; after 48-h of recovery, bAP rates were 4 and 6%
lower vs. BDC in DI and DI-TC, respectively (Figure 3).
Similarly, in the two groups, total intact OC level was maintained
during the first 48 h of dry immersion; then, a decrease was recorded
such as, at DI
120h
, the concentrations were 15% lower than BDC
values. 48 h of recovery are insufficient to recover the baseline levels
tending to keep concentrations at R
+48h
10% lower than BDC values
for both DI and DI-TC. Despite the lack of statistical significance,
Gla-OC evolved in a similar way to total intact OC for both groups.
No significant change was observed in its undercarboxylated forms
(Glu-OC) or in the Glu/Gla-OC ratio during all the study.
Osteocyte activity: Periostin, a gla domain vitamin K
dependent factor, is involved in many processes among which
the remodelling of bone tissue as a response to injury. In DI
group, circulating periostin concentrations progressively
decreased during all immersion period reaching, at DI
120h
,
levels equal to 82% of BDC rates; then these values returned
to baseline level after 48 h of recovery (Figure 3). When thigh cuff
was associated with dry immersion, a similar decrease than in DI
was observed, although not reaching statistical difference;
nevertheless, the decrease in periostin at DI
120h
was
significantly different in DI vs. DI-TC (p= 0.050).
Energy Metabolism and Hormones Levels
Visfatin concentrations increased as early as after 24 h of dry
immersion for the majority of subjects regardless of
treatment. It continued to increase throughout the dry
immersion phase until reaching, at DI
48h
, higher levels by
120 and 60% for DI and DI-TC respectively, when compared
to BDC (Figure 2); after 48 h of recovery, the level remained
above the BDC values for 7 of DI subjects whereas the
concentrations returned to or even decreased below the
basal values for DI-TC group. Furthermore, circulating
IGF1 levels progressively increased during immersion in
TABLE 2 | Body weight and VO
2max
before and at the end of dry immersion.
Variable Dry immersion (DI) Dry immersion with thigh cuffs (DI-TC)
BDC DI24h DI120h R+48h BDC DI24h DI120h R+48h
Morning weight, kg 73.5 72.6
a
71.8
a,b
72.8
a,b,d
80.2 78.7
a
78.3
a,b
79.4
a,d
(58.2–81.8)(63.5–87.7) (62.6–86.4) (62.6–86.4) (62.5–86,0) (59.3–82.9) (58.5–82.0) (58.0–81.1)
VO
2max
, ml/mn/kg 47.3 42.7
a
’46.9 43.1
a
(33.7–57.8) (31.8–53.4) (36.6–55.1) (37.2–49.0)
Measurements were made at baseline (BDC), during dry immersion (DI) and after recovery. Data are expressed as median (interquartile range) for n= 9 per group. a, b, d: significantly
different (pp<0.05) vs. BDC, DI24 h, DI120 h, respectively. a: trend to significance to BDC There was no difference between DI and DI-TC groups.
TABLE 3 | Serum markers for bone cellular activities in Dry Immersion (DI) and Dry Immersion with Thigh Cuffs (DI-TC) during the 5-day dry immersion and after 48 h-
recovery.
BDC DI
24h
DI
48h
DI
120h
R
+48h
Markers for bone turnover
Resorption activity
TRAP5b (U/L) DI 2.79(0.80–4.33) 3.12(0,93–4.88)
a
3.50(0.96–4.98)
a,b’
3.11(1.08–5.12)
a
3.44(1.05–4.94)
a
DI-TC 2.72(1.47–4.60) 2.93(1.80–6.60) 3.02(1.92–5.90)
a
3.05(1.90–5.89)
a
3.01(1.80–5.75)
CTx (pmol/L) DI 6229(1655–9831) 6259(1390–11012) 5977(1619–10180) 5811(1840–11746) 5977(2005–10546)
DI-TC 5638(2237–9460) 5966(2030–9436) 5830(1958–9780) 5480(1954–9505) 6613(2734–10839)
Formation activity
P1NP (µg/L) DI 67.2(37.7–109.6) 61.3(30.6–100.3)
a
59.8(35.0–103.1)
a
55.7(29.7–102.7)
a
58.6(32.8–94.9)
a,b
DI-TC 92.5(52.3–136.1) 84.1(45.5–114.7)
a
81.1(42.2–110.6)
a
68.9(41.4–110.6)
a
76.3(38.7–107.7)
a
bAP (µg/L) DI 16.0(10.8–20.7) 17.1(11.2–21.8)
a
17.6(12.8–25.7)
a,b’
16.5(10.4–22.1)
a,c
14.8(10.4–22.1)
b,c,d
DI-TC 21.4(9.0–27.3) 23.1(9.8–27.4)
a’
23.4(10.8–28.0)
a
22.4(9.9–27.7)
b’,c
20.2(8.6–25.3)
b,c,d
Intact OC (ng/ml) DI 23.7(17.9–39.3) 24.7(15.8–44.1) 24.2(17.6–44.7) 21.0(11.6–39.7)
a,b,c
21.2(14.8–39.3)
b,c
DI-TC 27.3(13.6–39.5) 26.6(12.3–39.9) 29.3(12.4–35.4) 23.3(9.9–30.4)
a,b,c
24.2(10.4–31.7)
a,b’,c
Gla-OC (ng/ml) DI 11.9(9.4–16.8) 13.3(9.4–18.0) 12.8(8.5–20.0) 11.8(6.5–18.8)
b,c
11.2(7.9–17.4)
a,b,c
DI-TC 12.0(7.3–15.8) 11.8(7.6–14.6) 11.9(7.8–16.2)
b
10.4(5.8–15.5)
c
11.3(6.6–15.5)
c
Glu-OC (ng/ml) DI 7.5(2.9–17.5) 7.6(3.7–15.5) 8.2(3.4–17.5) 5.7(2.9–16.7)
c
5.3(3.6–12.8)
b,c
DI-TC 7.6(4.3–16.3) 8.5(4.4–15.0) 9.3 (5.2–17.2)
b
7.3(3.4–11.8)
b’,c
6.9(3.5–13.1)
c
Osteocyte activity
Periostin (pmol/L) DI 990(615–1137) 934(577–1074) 708(592–990)
a,b
712(493–977)
a,b
904(710–1138)
c,d
DI-TC 740(518–1147) 835(631–1122) 729(508–1163) 742(505–1061) 713(589–1174)
Measurements were made at baseline (BDC), during dry immersion (DI) and after recovery. Data are expressed as median (interquartile range) for n= 9 per group. a,b,c,d: significantly
different (p<0.05) vs. BDC, DI
24h
,DI
48h
,DI
120h
, respectively. a’,b’: trend to significance (p≤0.07) vs. BDC, DI
24h
, respectively. There is no difference between DI and DI-TC groups.
TRAP5b: tartrate-resistant acid phosphatase isoform 5b; CTx: C-terminal crosslinked telopeptide of type I collagen; P1NP: procollagen type I N-terminal propeptide; bAP: bone alkaline
phosphatase; OC: osteocalcin; Gla-OCand Glu-OC: carboxylated and uncarboxylated osteocalcin, respectively.
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Linossier et al. DI-5-Cuffs and Bone Remodeling Markers
both groups until reaching, at DI
48h
,8and6%higher
concentrations for DI and DI-TC, respectively, when
compared to BDC ; levels remained elevated during all dry
immersion phase for DI group wheras, for DI-TC group,
concentrations returned to BDC level at DI
120h
;atthislatter
timing, IGF1 concentrations became significantly higher for
DI vs. DI-TC ; after 48-h of recovery, circulating IGF1 levels
were lower vs. BDC by 10 and 16% in DI and DI-TC,
respectively.
Lipocalin-2 and irisin, two potential regulators of bone
homeostasis, were unchanged for both groups during the
entire experiment.
TABLE 4 | Serum markers for phospho-calcic status and metabolic regulators in Dry Immersion (DI) and Dry Immersion with Thigh Cuffs (DI-TC) during the 5-day dry
immersion and after 48 h- recovery.
BDC DI
24 h
DI
48 h
DI
120 h
R
+
48 h
Markers for phospho-calcic metabolism
PTH (ng/L) DI 23.4(17.2–46.5) 28.0(16.0–40.5) 27.4(14.7–47.1) 21.2(14.5–33.4)
a’,b’
23.4(13.0–40.6)
b’
DI-TC 31.1(17.7–43.5) 31.2(19.0–44.2) 28.9(18.4–45.8) 27.5(20.2–35.7) 28.4(19.1–37.2)
Calcium (mg/L) DI 96.0(94.0–102.4) 100.0(95.6–105.6)
a
101.6(96.0–106.8)
a,b’
99.6(96.0–105.2)
a
96.4(90.8–100.4)
b,c,d
DI-TC 95.6(92.8–100.4) 96.8(94.4–102.8)
a,†
98.0(94.0–106.0)
a,†
96.4(92.4–100.8)* 92.8(90.8–98.8)
b,c,d
Phosphorus (mg/L) DI 38.1(32.6–49.9) 38.1(32.6–49.3) 37.8(33.8–48.1) 37.5(33.5–47.4) 36.9(32.9–47.4)
DI-TC 38.8(28.2–43.4) 38.1(30.1–47.1) 39.4(31.3–44.3) 37.8(29.5–45.6) 38.1(31.3–44.6)
Metabolism regulators
Visfatin (ng/ml) DI
n
0.85(0.22–1,52) 0.97(0.43–3.64)
a’
1.20(0.72–6.93) 1.98(0.66–2.90)
a’
1.21(0.13–2.62)
d’
DI-TC 0.79 (0.40–2.19) 1.73(0.42–3.32)
a’
1.06(0.43–4.58)
a’
2.11(0.37–3.79)
a
0.77(0.30–2.24)
b,c,d
Lipocalin-2 (ng/ml) DI 148(102–235) 148(102–224) 163(119–205) 161(115–237) 161(113–242)
DI-TC 173(113–191) 152(125–192) 159(126–193) 162(124–201) 164(120–194)
IGF1 (µg/L) DI 223(168–301) 225(189–292) 239(208–323)
a,b
246(181–318)
a
194(144–275)
a,b,c,d
DI-TC 220(141–242) 232(128–245)
a’
231(132–252)
a’
208(114–250)
b’,c, *
172(114–205)
a,b,c,d
Irisin (µg/ml) DI 6.0(2.7–15.2) 7.0(4.1–12.7) 6.0(2.9–15.7) 6.2(3.2–13.4) 5.7(2.6–13.5)
DI-TC 5.0(3.0–9.4) 5.3(3.7–10.2) 5.3(3.4–9.8) 4.1(2.9–8.4) 4.3(2.6–11.3)
Measurements were made at baseline (BDC), during dry immersion (DI) and after recovery. Data are expressed as median (interquartile range) for n= 9 per group, except for Visfatin
(
n
: analysis made for n = 8 only for DI group). a,b,c,d: significantly different (p<0.05) vs. BDC, DI
24h
,DI
48h
, and DI
120h
, respectively ; a’,b’,c’,d’: trend to significance (p<0.07) vs. BDC,
DI
24h
,DI
48h
, and DI
120h
, respectively ; †and *: trend (p<0.10) and significant difference (p<0.05) vs. DI group, respectively. PTH: parathyroid hormone; IGF1: insulin growth factor 1.
FIGURE 2 | Effect of Thigh cuff on dry immersion-induced changes on the metabolic response. At Top, changes in calcic metabolism. At bottom ,cha nges in values
of IGF1 and visfatin. Values are medians ± interquartile range for n= 9, except for Visfatin (analysis made for n= 8 only for DI group). a,b,c,d indicate significant differences
vs. BDC, DI
24h
,DI
48h
and DI
120h
, respectively. a’: trend to significance (p<0.10) vs. BDC. δindicate significant differences (p≤0.05) between DI and DI-TC groups.
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Linossier et al. DI-5-Cuffs and Bone Remodeling Markers
Associations Between Biochemical Blood
Markers
At baseline,significant correlations were found between bone
resorption markers (r = 0.785, p= 0.0001 between CTx and
Trap5b) and bone formation markers (r = 0.811, p<0.0001
between intact OC and P1NP). In addition, collagen bone
formation and mineralisation processes were also coupled (r =
0.683, p= 0.002 between P1NP and bAP). Moreover, statistical
analysis elucidated the coupling between resorption and
formation as shown by the relationships between intact OC
vs. CTx and Trap5b (r = 0.793, p<0.0001 and r = 0.669, p=
0.002, respectively) or between P1NP vs. CTx and Trap5b (r =
0.606, p= 0.008 and r = 0.650, p= 0.004, respectively).
Furthermore, Glu-OC concentrations were linked to those
of CTx and intact OC levels (r = 0.577, p=0.012andr=
0.666, p= 0.0026, respectively).
Changes in parameters at DI
48h
and/or DI
120h
: Since thigh
cuff had practically no significant effects on the measured
parameters, relationships between their changes were
established from all subjects (with or without thigh cuff i.e., 17
or 18 subjects) due to the dry immersion effect; they were
summarized in Table 5.
Whatever the duration of dry immersion, no correlation was
found between changes in CTx and Trap5b.
In the early dry immersion phase (at DI48 h exclusively), the
following relationships were obtained: 1) ΔGlu-OC positively
with ΔIGF1; 2) ΔVisfatin positively with ΔCalcium but negatively
with ΔTrap5b and ΔPhosphorus; 3) ΔP1NP positively with
ΔCalcium but negatively with ΔPhosphorus; 4) ΔCalcium
negatively with ΔPTH.
In the later dry immersion phase (at DI120 h), ΔGla-OC has
been correlated positively with ΔP1NP, ΔintactOC and ΔGlu-OC
but negatively with ΔPTH; In the same time, ΔCTx with
ΔPhosphorus, ΔIrisine with both ΔCTx, ΔPhosphorus and
ΔPeriostin; ΔbAP positively with ΔTrap5b but negatively with
ΔPeriostin; ΔCalcium with ΔIGF1; ΔGlu-OC with ΔTrap5b.
FIGURE 3 | Effect of Thigh cuff on dry immersion-induced changes on bone metabolism. Changes in bone resorption and formation or mineralisation markers
during dry immersion without (white) or with (grey) thigh cuff. Values are expressed as medians ± interquartile range for n= 9 per group. a,b,c,d indicate significant
differences vs. BDC, DI
24h
,DI
48h
and DI
120h
, respectively. a’indicate trend to significance vs. BDC. δ‘and δindicate trend (p<0.10) or significant differences (p≤0.05)
between DI and DI-TC groups. TRAP5b: tartrate-resistant acid phosphatase isoform 5b; P1NP: procollagen type I N-terminal propeptide; bAP: bone alkaline
phosphatase; intact OC: intact osteocalcin.
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Linossier et al. DI-5-Cuffs and Bone Remodeling Markers
TABLE 5 | Association between biochemical blood markers.
Markers Trap5b CTx Glu-
OC
bAP P1NP Int
OC
Gla-
OC
Periostin IGF1 Visfatin Calcium PTH Phosphorus Irisin
Trap5b BDC
value
0.79 - - 0.65 0.67 - - - - - - - -
Δat
DI48h
---- - - - -−0.50 - - - -
Δat
DI120h
- 0.61 ** 0.62 - - - - - - - - - -
CTx BDC
value
0.58 * - 0.61** 0.79 - - - - - - - -
Δat
DI48h
0.58 * - - - - - - - - - - -
Δat
DI120h
0.59 ** - - - - - - - - - 0.48 * 0.54 *
Glu-OC BDC
value
- - 0.67** - - - - - - - -
Δat
DI48h
- - 0.48* - - 0.52 * - - - - -
Δat
DI120h
- - 0.52 * 0.53 * - - - - - - -
bAP BDC
value
0.68 - - - - - - - - -
Δat
DI48h
- - - - 0.56 * - - - - -
Δat
DI120h
-- -−0.60 ** 0.51 * - - - - -
P1NP BDC
value
0.81 - - - - - - - -
Δat
DI48h
0.52* - - - - 0.58 * - −0.50 * -
Δat
DI120h
0.51 * 0.65** - - - - - - -
int OC BDC
value
------ - -
Δat
DI48h
------ - -
Δat
DI120h
0.71 ** - - - - - - -
Gla-OC BDC
value
--- -- - -
Δat
DI48h
--- -- - -
Δat
DI120h
--- -−0.48 - -
Periostin BDC
value
-- - - - -
Δat
DI48h
-- - - - -
Δat
DI120h
-- - - -0.54*
IGF1 BDC
value
--- - -
Δat
DI48h
--- - -
Δat
DI120h
- 0.61 ** - - -
Visfatin BDC
value
-- - -
Δat
DI48h
0.52 * - −0.52 * -
Δat
DI120h
-- - -
(Continued on following page)
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Linossier et al. DI-5-Cuffs and Bone Remodeling Markers
After both 48 and 120 h of dry immersion, changes in P1NP
and intact OC were positively correlated. It is also worth noting
the positive relationships between: 1) ΔGlu-OC (decarboxylated
OC form) and both Δintact OC and ΔCTx; 2) ΔbAP and ΔIGF1.
DISCUSSION
Our current results confirmed that the microgravity simulated by
short-term DI model challenges bone remodelling activity in
human (Linossier et al., 2017). It demonstrated rapid bone
adaptation with a metabolic peak at 48 h of DI followed by
the maintenance of a dissociation between bone formation and
resorption beyond the end of the dry immersion conditions.
Despite beneficial effects induced by thigh cuff on body fluid
changes (Robin et al., 2020), this countermeasure did not prevent
the deleterious effects in bone cellular activities.
Dry Immersion Conditions and Bone
Remodelling Activity
As early as 24-h DI, resorption evaluated from circulating Trap5b
level was already elevated by 7%. After 48 h of immersion, this
marker continues to increase up to 14%, which is greater than the
7% observed at the same time in the first experiment of 3 days
(Linossier et al., 2017). Regarding bone formation activity, P1NP
concentrations indicative of lower type 1 collagen deposition
decreased while bAP, an enzyme promoting bone mineralisation,
increased, as also observed at DI
48h
in the shorter 3-d study. Our
current results confirmed that the microgravity simulated model
by short-term DI challenges bone remodelling activity.
Sclerostin, an osteocyte-secreted protein negatively regulating
osteoblast activity and therefore bone formation, has been shown
to increase from 8 to 10 days of bedrest (Frings-Meuthen et al.,
2013) suggesting that it is a late responding marker. In line with
these results, we did not detect any change in the 3-day dry
immersion experiment (Linossier et al., 2017) and did not
analyzed it in the present study. However, we assessed
periostin, a matricellular protein of cortical bone and
periosteum and found that its serum level decreased as early
as 48 h in DI group. During hindlimb suspension in mice
periostin expression decreased and contributed to cortical
bone loss through an increase in sclerostin (Gerbaix et al.,
2015). Periostin-deficient mice have low bone mass and
respond less to physical activity due to a lack of sclerostin
inhibition by mechanical loading (Bonnet et al., 2009,2012).
After 4.5–6-months spaceflight in cosmonauts, periostin serum
level was showed to predict tibia cortical evolution during space
mission (Vico et al., 2017). In the current 5-d DI, periostin drop
may thus be indicative of a later reduction in its antisclerostin
activity.
Extending the dry immersion from 3 to 5-days revealed
adaptation kinetics with bone marker changes for Trap5b,
bAP, P1NP and periostin reaching a peak at 48 h of DI
followed by stabilization at this new steady-state throughout
unloading condition. The DI-induced alteration in bone
TABLE 5 | (Continued) Association between biochemical blood markers.
Markers Trap5b CTx Glu-
OC
bAP P1NP Int
OC
Gla-
OC
Periostin IGF1 Visfatin Calcium PTH Phosphorus Irisin
Calcium BDC
value
---
Δat
DI48h
−0.60 - -
Δat
DI120h
---
PTH BDC
value
--
Δat
DI48h
--
Δat
DI120h
--
Phosphorus BDC
value
-
Δat
DI48h
-
Δat
DI120h
0.52 *
Irisine BDC
value
Δat
DI48h
Δat
DI120h
Spearman correlation coefficients between values at BDC or between percent changes at DI
48h
or DI
120h
in bone and energy metabolism parameters for n= 18 except for Visfatin which
was made for n= 17. Changes are expressed in percent from BDC. The values of Spearman r wereindexed by the significance (*, ** or *** for p value <0.05, 0.01 or 0.001, respectively).
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Linossier et al. DI-5-Cuffs and Bone Remodeling Markers
formation activity was confirmed by the significant drop in total
OC and its carboxylated form (Gla-OC) at DI
120h
. Changes in
bone formation markers (P1NP, OC, and Gla-OC) were
correlated with each other. The evolution of total OC seems to
result also from the change in its decarboxylated forms: when
considering the 18 subjects together, whereas Glu-OC levels were
found higher at DI
48h
, they decreased significantly after 5 days of
DI, as did total OC.
This dissociation between bone cellular activities supports the
rapid onset of bone alterations. Serum markers give a global trend
that might result from local changes as evidenced by some authors
who reported, as early as 7 days of DI, a decrease in bone mineral
density of the lower part of the skeleton (proximal epiphysis of the
femur) to the benefit of the upper part (skull, hand, costal bones)
when compared to baseline (Kotov et al., 2003).
Dissociation of Serum Markers Within the
Activities of Either Bone Formation or Bone
Resorption
Regarding the bone formation markers and as in the previous 3-
day campain, we observed a dissociation between the evolution of
bone matrix synthesis and mineralisation markers. Indeed, serum
P1NP and OC decreased whereas serum levels of bAP increased
in the two groups. While, at baseline, these processes were quite
well coupled as evidenced by the correlation established between
P1NP and bAP, DI conditions would seem to have induced a
decoupling. It would be interesting to see if such an adaptation
exists in other unloading models.
Regarding the bone resorption markers and, contrary to the
previous study, no change was observed in circulating CTx levels,
even after 5 days of DI. Several explanations could be at the origin
of the dissociation between changes in Trap5b and CTx. First,
contrary to CTx, the Trap5b level is independent of renal function
as it is rapidly inactivated during circulation by the loss of iron
(Halleen et al., 2000). Renal function is altered by DI as evidenced
by increased renal excretion of liquids and negative water balance
(Larina and Kysto, 2008; Navasiolava et al., 2011; Noskov et al.,
2011). This was confirmed in our subjects whose results were
published previously by Robin et al. (2020). Therefore, in such
situations, serum TRAP5b appeared to be a more relevant
indicator than telopeptides of type I collagen for the
estimation of bone resorption, as shown in hemodialysis
patients (Shidara et al., 2008). Second, this dissociation
between TRAP5b and CTx could be a timing issue. TRAP5b
levels reflect the osteoclast numbers while CTx is released into the
circulation in response to osteoclast function (Henriksen et al.,
2007). At the basal level, our subjects were characterized by a 24%
lower Trap5b activity when compared to the 1st experiment (p<
0.05) leading to suppose a lower number of osteoclasts whereas
CTx levels were similar in the two studies. Therefore, it is possible
that higher TRAP5b increases induced by DI conditions, bringing
circulating levels at 48-h DI similar in the 2 studies, preceded
those of CTx. For continuous bone remodelling and tissue
reparation of the skeleton, the number of osteoclasts rather
than their functional activity appeared essential to mediate the
coupling between bone resorption and bone formation (Schaller
et al., 2004;Karsdal et al., 2005;Koh et al., 2005;Martin and Sims,
2005). When we considered changes at DI
120h
for all 18 subjects
together, we found some relationships that seemed to agree with
these latter ones. Indeed, changes in Trap5b are positively
correlated with those of the mineralisation marker (bAP) and
the decarboxylated OC form (Glu-OC). Despite the lack of
significant variation in both CTx and Glu-OC in DI group,
these 2 markers remain correlated whether on baseline or in
terms of variations at both DI
48h
and DI
120h,
supporting a role for
bone resorption in the decarboxylation of osteocalcin.
Relationship Between Bone Remodelling
and Associated Metabolic Responses
In our previous study, the increased resorption had been
associated with the onset of insulin resistance along with a
higher degree of OC decarboxylation and a higher circulating
IGF1 (Linossier et al., 2017). In the present study, blood glucose
was unmodified (Robin et al., 2020), but data on blood insulin are
lacking. On the other hand, circulating IGF1 levels were increased
by 8% for the DI group at DI
48h
, which could be the direct
consequence of the stimulated GH secretion following a potential
increase of the insulin response. When compared to the first dry
immersion experiment, this activation was shifted by 24-h (IGF1
levels not yet increased at DI
24h
) and was less than the 15% higher
IGF1 concentration observed at DI
48h
in our previous study.
Moreover, this could not be associated with a significant increase
in degree of OC decarboxylation although 8 out of 9 subjects
increased their Glu-OC concentration at DI
48h
in DI group.
Despite the small sample size, we showed that the higher the
rates of IGF1, the higher the circulating rates of Glu-OC (r = 0.70,
p= 0.0358 at DI
48h
for DI group). One of the rare studies which
have measured Glu-OC under unloading models (Morgan et al.,
1985) reported no change while bone resorption activity was
increased by 80% after 30 days of HDBR.
IGF1is known to stimulate bone formation. However, as
already shown in Linossier et al. (2017), increased circulating
IGF1 levels were associated with decreased bone formation
markers under dry immersion. The present study is in line
with Bikle and colleagues as well as Long and colleagues (Bikle
et al., 1994,2015;Long et al., 2011) showing that unloading would
induce the development of resistance to this growth factor.
Indeed, in rats submitted to hindlimb unloading (Bikle et al.,
1994) observed the failure of the unloaded bone to grow in
response to exogenous IGF1. In the same model, Long et al.
(2011) showed that bone marrow osteoprogenitor cells
originating from unloaded rats failed to respond to IGF-1
treatment in vitro.
Comparison Between the Current 5-d dry
Immersion and a Previous 3-d dry
Immersion Might Explain Some Differences
in Serum Marker Changes
As demonstrated for the first time during the previous 3-day dry
immersion experiment, our results confirmed the great
responsiveness for visfatin. However,asforIGF1,thevisfatin
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Linossier et al. DI-5-Cuffs and Bone Remodeling Markers
increase was delayed by 24 h (Linossier et al., 2017). This shift in the
timingofthemetabolicresponsemightberelatedtothedifferencein
training level of the subjects between these two studies. We
compared the characteristics of our subjects with those of the
group from the first experiment published in 2017. It was found
that, while no difference in baseline group characteristics related to
age, height, weight and BMI were reported between these two
studies, VO
2max
was found to be 20% higher in the present study
as compared to the previous one (p<0.05). This better aerobic
capacity was accompanied by visfatin rates 4 to 5 times lower at
BDC
-1
(p<0.0001) when compared to the first experiment. In bed
rest studies that consider alterations in bone cellular activities, no
VO
2max
data were reported. The value, reported elsewhere in HDBR
studies (Capelli et al., 2006;Bringard et al., 2010), are comparable to
those of the subjects of our first experiment. The VO
2max
level of the
subjects in this 2
nd
experiment remains higher than in all these
studies. Rare are studies that explored relationship between physical
ability and visfatin levels in healthy men. Choi et al. (2007) reported
that aerobic exercise training induced a decrease in plasma visfatin
levels in Korean women. The lack of a consensus on the normal
visfatin concentration and its relationship to anthropometric and
metabolic parameters in adult has led Jurdana et al. (2013)) to study
the association between increased circulating visfatin levels and
anthropometric parameters in obesity. These authors evidenced
that physical fitness was the best significant predictor of baseline
visfatin concentration in male participants. Such observations could
explain that, at BDC
-1
, visfatin levels were much lower for DI and
DI-TC groups when compared with our previous study.
Furthermore, when compared to the 1st experiment, the 18
subjects were characterized not only by a 24% lower Trap5b
activity (p<0.05) but also by a 22% higher osteocalcin level
(p<0.05) at BDC
-1
; this evidenced a different bone remodelling
rate with lower bone resorption and higher bone formation activities
in the present study when compared to 3-day DI experiment. Such
differences are apparently due to better physical ability. In line with
this statement (Gabel et al., 2021) recently reported in astronauts
that pre-flight markers of bone turnover and exercise history may
identify crewmembers at greatest risk of bone loss due to unloading.
Reversibility of Dry Immersion-Induced
Changes
Similar to bone cellular activities, associated metabolic
processes responded with an early phase of adaptation with
a peak at 48 h of DI that maintains at the new steady-state
throughout unloading condition. Adaptation processes to
unloading are partially reversible since, after 48 h of
recovery, only some of the markers (i.e., Ca, PTH, bAP,
periostin and visfatin) returned to their basal level. Switch
from unloaded condition to 1g is associated with a persistent
impairment of bone turnover markers (Trap5b, P1NP and OC
and its forms) as well as a significant decline in circulating
IGF1 concentrations below the basal level. The endocrine role
of bone could again be responsible for the production of IGF1
below BDC as a result of reduced stimulation of Langerhans
cells due to lower circulating Glu-OC levels.
Do Thigh Cuffs Prevent the Early dry
Immersion Induced Effects on Bone
Remodelling Activity and Energy
Metabolism: dry Immersion-TC vs. dry
Immersion Group?
The effects of venoconstrictive thigh cuffs on body fluid changes
from the subjects of this experiment were recently reported by
Robin et al. (2020). These authors evidenced that the use of thigh
cuffs was associated with a slowdown and limitation of the total
body water loss and a trend to limit the decrease in plasma
volume. Nevertheless, these beneficial effects of thigh cuff on
body fluid changes remained very moderate and did not
counteract decreased tolerance to orthostatic challenge. No
study has considered the impact of thigh cuff on the bone
cellular activities and muscle deconditioning induced by real
or simulated microgravity. Here, thigh cuff is ineffective since
metabolic profiles of bone resorption and formation remained
similar between the two groups. At DI
48h
, the trends towards a
lesser increase in bAP and a lower decrease in P1NP as well as a
lower release of calcium under thigh cuff were probably more
related to the limitation of plasma volume loss by 1/4–1/3 under
thigh cuffs when compared to immersion alone (Robin et al.,
2020). On the other hand, it is worth noting the absence of a
significant decrease in periostin contrary to DI condition only
under thigh cuffs. The application of thigh cuffs might have been
more effective on energy metabolism in partially preventing the
IGF1 stimulation and visfatin production. However, as for
previous markers, differences in IGF1 being minimal between
the two groups should be considered with great care. On the other
hand, the difference in plasma volume cannot by itself explain the
lower (but not significant) percentage increase in visfatin levels
under thigh cuff vs. DI. (60 vs. 120%).
Limitations
The main limitation of this study is the low number of subjects in
each group (n= 9). The number of subjects was not specifically
powered for bone metabolism criteria, but for change in optic
nerve sheath diameter, a primary outcome measure for this
protocol (ck Kermorgant et al., 2021). This might have been
responsible for the lack of significant changes, as seen for example
in Glu-OC since statistical difference was achieved with the
unique group of 12 subjects in the 1st experiment (Linossier
et al., 2017). Also, due to the numerous teams involved in this
experiment, limited serum was available which prevented us from
testing more parameters (such as sclerostin, markers of insulinic
response or renal function).
CONCLUSION
The present study confirmed the early adaptation of bone
activities and increased visfatin levels under dry immersion
conditions. It also allowed the possibility to substantiate the
dissociation between bone resorption and formation, which
was maintained throughout the immersion period with a
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Linossier et al. DI-5-Cuffs and Bone Remodeling Markers
metabolic peak at 48 hours and was not restored 48 h after the
reloading. The disconnection observed between bone collagen
synthesis and its mineralisation highlighted dysfunction in the
bone formation phase. In view of the changes in fluid-volume
homeostasis, it seemed relevant to question the impact of fluid
transfers on the bone metabolism. However, thigh cuffs applied
intermittently to reproduce in-flight use and avoid deleterious
venous effects did not allow to prevent the early dry immersion
induced effects on bone remodelling activity and energy
metabolism. This suggests that body fluids modifications are
not the leading events for bone changes under DI. Another DI
effects, not counteracted by thigh cuffs, such as supportlessness,
mechanical and axial unloading, and physical inactivity,
seemingly dominate for bone and metabolic responses. This
trial is the second DI study done in Europe and we believe
that the data obtained are valuable to set up effective
countermeasures for the effects of gravitational unloading.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/Supplementary Material, further inquiries can be
directed to the corresponding author.
ETHICS STATEMENT
The studies involving human participants were reviewed and
approved by local Ethic Committee (CPP Est III: October 2,
2018, n°ID RCB 2018-A01470-55) and French Health
Authorities (ANSM: August 13, 2018). ClinicalTrials.gov
Identifier: NCT03915457. The patients/participants
provided their written informed consent to participate in
this study.
AUTHOR CONTRIBUTIONS
Conception and research design: M-PB, AB, and GG-K.
Experimental work and data analysis: M-TL, LP, MN, and CB.
Analysis and interpretation of results: M-TL and LV. Drafting
and revising the manuscript: M-TL, LV, LP, and PF.
FUNDING
This dry immersion protocol was supported by CNES (DAR
CNES N◦2018 –4800000970).
ACKNOWLEDGMENTS
We thank the volunteers, the staff of MEDES for the participation
in this protocol at the MEDES space clinic in 2018–2019, and the
Biochemistry lab (Saint-Etienne University Hospital) for bone
marker analyses.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fphys.2022.801448/
full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
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Frontiers in Physiology | www.frontiersin.org April 2022 | Volume 13 | Article 80144813
Linossier et al. DI-5-Cuffs and Bone Remodeling Markers
