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. 2025 Aug 15;26:101871. doi: 10.1016/j.bonr.2025.101871

Simulated microgravity accurately models long-duration spaceflight effects on bone and skeletal muscle in skeletally immature mice☆

Michael A Friedman a, Yasmina Zeineddine a,b, Olivier Tuyambaze a, Wesam Elhawabri a, Ahmed Al Shammary a, Louis Stodieck c,d, Virginia L Ferguson c,d, Henry J Donahue a,⁎
PMCID: PMC12398213  PMID: 40894391

Abstract

Spaceflight (SF) and disuse result in decreases in bone and skeletal muscle volume that increase fracture risk. Hindlimb unloading (HLU) has been widely used to model the effects of microgravity. However, the effects of SF and HLU on bone and skeletal muscle have not been directly compared during long-duration SF. We examined the effects of five weeks of SF and HLU in the femurs of female Balb/c mice. For the first time, SF and HLU were directly compared using mice of the same age, strain, sex, and duration as a mission to the ISS. We hypothesized that HLU would accurately model SF, resulting in similar bone and skeletal muscle loss. Ten-week old female Balb/c mice were assigned to baseline, vivarium control, habitat control, and SF groups (n = 10/group). A separate cohort of 10-week female Balb/c mice were placed in HLU or control (n = 10/group). Femoral cortical area increased from baseline in all groups except HLU. The magnitudes of increases were lower in the SF and HLU groups. Similar effects were seen in trabecular bone. Femoral ultimate force decreased in SF and HLU groups, compared to control groups. Gastrocnemius and quadriceps mass was lower in SF and HLU mice than in control mice. HLU resulted in greater bone loss than SF, possibly due to differences in housing conditions. HLU effectively models long-duration effects of SF on the musculoskeletal system, highlighting its utility for studying astronaut health risks and developing countermeasures.

Keywords: Spaceflight, Bone, Skeletal muscle, Animal model, Hindlimb unloading

Highlights

  • •

    Hindlimb unloading models long-duration spaceflight effects on bone and skeletal muscle mass in growing mice.

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    Cage style impacts bone and skeletal muscle, highlighting the need to match cages in spaceflight and ground controls.

  • •

    Spaceflight and hindlimb unloading limit cortical bone mass and bone strength gains during skeletal development.

1. Introduction

Spaceflight (SF) and disuse result in decreases in bone and skeletal muscle volume that decrease bone and muscle strength, increasing fracture risk (Lang et al., 2004; Man et al., 2022; Juhl et al., 2021; Coulombe et al., 2020). A meta-analysis of rodents in SF found rodents lose around 1.7 % of trabecular bone volume per day, raising serious concerns for long-duration spaceflight missions (Fu et al., 2021). Bone loss typically occurs through a reduction in bone formation in young, skeletally immature mice and an increase in bone resorption in skeletally mature adult mice (Fu et al., 2021; Coulombe et al., 2021). Muscle atrophy occurs through a reduction in protein synthesis and an increase in protein degradation (Lee et al., 2022). These changes are accompanied by a shift in muscle fiber size and fiber type (Fitts et al., 2010). Countermeasures are needed to prevent bone and skeletal muscle loss during long-duration SF to ensure astronaut safety and mission success.

Animal research in space is costly, and sample sizes and flight opportunities are limited. For more efficient screening of mechanisms involved in unloading, animal models of SF are used on Earth. The most widely used model, hindlimb unloading (HLU), has been used for many years to model the effects of microgravity (Globus and Morey-Holton, 2016). HLU involves suspending mice by their tails at an angle that prevents their hindlimbs from touching the ground. The cages have a wire bottom floor that mice grab with their forelimbs to move and access food and water. Compared to other models of unloading, HLU best models the effects of microgravity by inducing extensive musculoskeletal atrophy and cephalad fluid shift.

Typically, HLU is administered for 1–3 weeks to study acute effects of unloading. This duration mirrors that of SF studies performed during shorter missions prior to the creation of the International Space Station (ISS) research lab. Animal studies on the ISS have been performed for longer durations of 1–2 months to study the effects of long-duration spaceflight. However, scarce literature exists on long-duration HLU. It remains unclear how accurately HLU models long-term effects of SF on bone and skeletal muscle.

As future space missions to the moon and Mars will require long-duration SF with a more diverse crew, we examined the effects of 6 weeks SF and HLU in skeletally immature, 10-week old female Balb/c mice. Studies of growing mice and rats have shown SF and HLU reduce bone formation and increase bone resorption, resulting in decreased bone volume (Coulombe et al., 2021; Gamboa et al., 2021; Gao et al., 2024). However, SF and HLU studies are not run in parallel. Typically, SF and HLU studies have mice of different age, strain, sex, and study duration, making direct comparison of SF and HLU results difficult. To address this issue, in this study, SF and HLU were directly compared using parallel experiments with mice of the same age, strain, sex, and duration as a mission to the ISS. We hypothesized that both long-duration SF and HLU would induce catabolic effects on bone volume, bone strength, and muscle mass, with no significant differences between the two models.

2. Methods

2.1. Experimental groups

All animal procedures were done with the approval of the NASA IACUC (#FLT-18-116) and Virginia Commonwealth University IACUC (Protocol #AD10001341). Female Balb/cAnNTac Murine Pathogen Free™ mice were ordered from Taconic Farms at 8 weeks of age. All experimental procedures began when the mice were 10 weeks of age. Mice were randomly assigned to six groups (n = 10 mice per group): Baseline, Vivarium Control, Habitat Control, Spaceflight (SF), Hindlimb Unloading Control (HLU Control), or Hindlimb Unloading (HLU).

2.2. Spaceflight model

Mice from the Baseline, Vivarium Control, Habitat Control, and Spaceflight (SF) groups were part of the Center for Advancement of Science in Space (CASIS) Rodent Reference Research Mission 1 (RRRM-1), NASA Rodent Research-8 (RR-8) experiment. All mice were initially housed at Kennedy Space Center (KSC). All mice were acclimated to standard cages with wire mesh floors and provided with NASA Rodent Nutrient Food Bars and water bottles equipped with lixits, matching those used in the NASA Rodent Habitats. Baseline mice were sacrificed at the time of launch (10 weeks of age). Habitat Control and SF mice were housed in NASA Rodent Habitats, which are cages specifically designed for spaceflight. SF mice spent 39.5 days in space, after which they returned to Earth alive. Two days after splashdown, SF mice were transported to The Scripps Research Institute (TSRI), where they were sacrificed, and tissues were harvested. Vivarium Control and Habitat Control mice remained at KSC during the flight period. Approximately one week prior to sacrifice, both groups were shipped in an environmentally controlled transport vehicle to TSRI, where they were acclimated and then sacrificed. Tissues from all groups were harvested at TSRI using identical protocols. Mouse femurs were stored in neutral buffered PBS at −20 °C. Muscle samples were stored in RNA Later overnight and then transferred to −80 °C for storage.

2.3. Hindlimb unloading model

Experimental procedures involving HLU Control and HLU mice were performed at Virginia Commonwealth University. Mice were housed two per cage and had access to Teklad LM-485 chow (Envigo) and water ad libitum. One week prior to the start of the experiment, HLU Control and HLU mice were placed in wire bottom rat cages to acclimate (Friedman et al., 2023; Buettmann et al., 2023). Two mice were placed in each cage. At the start of the experiment, mice were suspended by attaching their tails to a bar that was placed near the top of the cage. The mice had their tails held at a 30° angle, and they were monitored daily for health issues and to ensure they remained suspended at the same angle. While in suspension, mice could use their forelimbs to hold the wire bottom floor of the cage and move around the cage. Two mice were placed in suspension in each cage, facing away from each other and unable to make contact with each other. HLU controls were placed two per cage in the same style of cage, but they had no restraints placed on them and were free to roam around the cage. Mice were kept in HLU for 5 weeks (35 days). Then, mice were returned to normal ambulation for 24 h, to model the brief re-exposure to gravity that SF mice experienced when they were returned to Earth alive.

2.4. Micro-CT analysis of bone morphology

Mouse left femurs from HLU controls and HLU mice were scanned by in vivo micro-CT (Bruker Skyscan 1276) on day 1 and on the final day of the experiments. Left femurs from all other groups were scanned ex vivo in the same scanner. Scanning was performed using a 7.0 μm voxel size, with 60 kV, 200 μA, 730 ms exposure, and a 0.5 mm Al filter. All bones were reoriented along anatomical landmarks. Femur morphology was evaluated at the mid-diaphysis, the distal metaphysis, and the distal epiphysis using CTAn. A 180 μm region of interest of the mid-diaphysis located 4 mm distal to the lesser trochanter was used for cortical bone measurements. A 750 μm region of interest of the distal metaphysis located 200 μm proximal to the growth plate was used for metaphyseal trabecular bone measurements. A 520 μm region of interest of the distal epiphysis located immediately distal to the growth plate was used for epiphyseal trabecular bone measurements.

2.5. Mechanical testing of femur

Femur mechanical properties were measured by three-point bending to failure, as we have described previously (Friedman et al., 2023). Bones were loaded at 1.0 mm/min, with the anterior side in tension, using support spans of 6 mm. This span length was chosen to accommodate bone morphology from all 4 groups. The location of the fracture site was measured using calipers. Then micro-CT measurements of bone morphology at the fracture site were used to estimate tissue-level mechanical properties (Turner and Burr, 1993).

2.6. Gene expression

After dissection, left tibia, gastrocnemius and quadriceps were stored in RNAlater (Thermo Fisher Scientific, Cleveland, OH) at 4 °C for 16 h. Then the RNAlater was removed from the tissue samples, and the samples were kept at −80 °C. RNA was extracted from the tissues using the Qiagen RNEasy fibrous tissue kit (Qiagen, Valencia, CA). There was insufficient RNA quantity from bone samples, so only gastrocnemius muscle gene expression was analyzed. All samples had candidate gene expression (ΔCT) measured by RT-qPCR (CFX96 Real-Time System, Bio-Rad, Hercules, CA). Genes were chosen based on previous literature demonstrating these genes were affected by SF (Smith et al., 2020; Hayashi et al., 2023; Bodine et al., 2001; Okada et al., 2021; Di Filippo et al., 2024; Hanson et al., 2013). The primers were purchased from Bio-Rad:

Cacng1 (ID: qMmuCID0005384, Chromosome location: 11:107704388-107716164), Col1a1 (ID: qMmuCID0021007, Chromosome location: 11:94936401-94937999), Eif4ebp1 (ID: qMmuCED0047679, Chromosome location: 8:27275226-27275326), Fbxo32 (ID: qMmuCID0011869, Chromosome location: 15:58184197-58191332), Fndc5 (ID: qMmuCID0011876, Chromosome location: 4:129139883-129142123), Mstn (ID: qMmuCID0019470, Chromosome location: 1:53062084-53063928), Myh4 (ID: qMmuCID0022344, Chromosome location: 11:67248693-67250004), Myh7 (ID: qMmuCID0027443, Chromosome location: 14:54980370-54982238), Myog (ID: qMmuCED0046470, Chromosome location: 1:134292061-134292209), and Trim63 (ID: qMmuCID0014591, Chromosome location: 4:134325651-134327772). Gene expression was normalized to the average expression of Ap3d1 (ID: qMmuCID0022103, Chromosome location: 10:80721616-80723589) and Rpl13a (ID: qMmuCED0040629, Chromosome location: 7:45125911-45126010) (Hildyard and Wells, 2014).

2.7. Muscle metabolism protein analysis

Proteins were isolated from quadriceps muscles and analyzed by Western blot as previously described (Zeineddine et al., 2023; Maroni et al., 2021). Briefly, 20 mg of tissue was homogenized, and protein concentration was quantified via BCA assay (Thermo Fisher Scientific). An equal amount (30 μg) of protein of each sample was added to wells of 4–20 % gels (Bio-Rad; Hercules, CA) and subjected them to SDS-PAGE. Antibodies for total and phosphorylated p70S6K1 (T389, Cell Signaling Technology; Boston, MA) and 4EBP1 (T37/46 Cell Signaling Technology). The primary antibodies were diluted 1:1000, and the secondary antibodies were diluted 1:3000. Bound immune complexes were detected with Western ECL Substrate (Bio-Rad). Bio-Rad Image Lab software (version 6.1) was used to quantify proteins, normalized to total protein. The ratio of phosphorylated protein to total protein was used for analyses.

2.8. Statistical analysis

Data were analyzed using Graphpad Prism version 10.4. Significance was defined as p < 0.05. Body weight was analyzed by two-way repeated measures ANOVA with initial and final body weight. Sidak's multiple comparisons tests were used post-hoc. Bone and skeletal muscle data were analyzed by one-way ANOVA, with Tukey's multiple comparisons tests done post-hoc. For datasets expressed as percent change from baseline, two different normalization methods were used: one based on each animal's individual baseline, and another relative to the average value of the Baseline group. These differing approaches resulted in unequal group variances. To accommodate this, Brown-Forsythe ANOVA was applied, followed by Dunnett's T3 multiple comparisons tests.

3. Results

3.1. Femur length increased from baseline in all groups. HLU mice had a lesser increase in femur length than HLU control mice

Mouse body weight increased significantly (p < 0.05) over the ∼39 day treatment period from baseline to sacrifice in all control groups, but not in the SF and HLU groups (Table 1). Femur length was significantly greater in Vivarium Control mice compared to Baseline mice (Fig. 1A). No other groups showed a significant difference in femur length compared to Baseline mice. For Vivarium Control, Habitat Control, and SF mice, the percent change in femur length from baseline was calculated using the mean femur length of Baseline mice as the reference value (Fig. 2B). For HLU Control and HLU mice, we used longitudinal CT scans to measure the percent change from baseline for femur length. All groups underwent treatment periods, 39.5 days for the SF and corresponding control groups and 35 days for the HLU and HLU Control groups, and showed increased femur length from baselines, as expected for skeletally immature mice. While HLU mice had significantly less femur growth than HLU Control mice, no significant changes in femur length were observed in spaceflight.

Table 1.

Body weight at initial (10 weeks old) and at the final experimental endpoint (10 weeks old for baseline, 15 weeks old for all other groups) (mean ± standard deviation). *p < 0.05

Group Initial BW (g) Final BW (g) Change (p-value)
Baseline 19.51 ± 0.87 19.98 ± 0.80 *0.041
Vivarium control 20.27 ± 1.53 22.11 ± 1.55 *5.39E−04
Habitat control 20.33 ± 1.40 21.90 ± 1.20 *4.17E−04
Spaceflight 19.81 ± 1.54 19.80 ± 1.18 0.966
Hindlimb unloading control 18.83 ± 2.11 21.61 ± 1.20 *2.66E−03
Hindlimb unloading 18.69 ± 1.81 18.99 ± 1.43 0.487
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Fig. 1.

Fig. 1

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Left femur length and mid-diaphyseal cortical area and cortical area fraction (mean ± standard deviation). Unloading from spaceflight or hindlimb unloading (HLU) attenuated bone growth, resulting in lower femur length and cortical area. Data shown as final measurements after 6 weeks of spaceflight or hindlimb unloading and percent change from baseline. Groups that share individual data point symbol style had the same mouse cage style. For percent change from baseline, Vivarium Control, Habitat Control, and Spaceflight were normalized by the mean of the Baseline group. HLU control and HLU were normalized by in vivo CT scans of the same mice on day 1. Groups that do not share a letter were significantly different (p < 0.05).

Fig. 2.

Fig. 2

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Left femur distal epiphyseal bone volume fraction and distal metaphyseal bone volume fraction (mean ± standard deviation). Unloading from spaceflight or hindlimb unloading (HLU) resulted in greater decreases in epiphyseal BV/TV than Habitat Control and HLU Control mice, respectively. Data shown as final measurements after 6 weeks of spaceflight or hindlimb unloading and percent change from baseline. Groups that share individual data point symbol style had the same mouse cage style. For percent change from baseline, Vivarium Control, Habitat Control, and Spaceflight were normalized by the mean of the Baseline group. HLU control and HLU were normalized by in vivo CT scans of the same mice on day 1. Groups that do not share a letter were significantly different (p < 0.05).

3.2. SF and HLU mice had lower femur cortical area than their respective controls. HLU mice had lower femur cortical area than SF mice

Cortical area and cortical area fraction were significantly greater in Vivarium Control and Habitat Control mice than in Baseline mice (Fig. 1C, 1E). Cortical area and cortical area fraction were significantly lower in HLU mice than in HLU Control mice. SF mice had significantly lower cortical area than Vivarium Control mice. Interestingly, HLU mice had significantly lower cortical area and cortical area fraction than SF mice. All groups had an increase in cortical area and cortical area fraction from baseline, except HLU mice (Fig. 1D, 1F). Additionally, SF mice had significantly less increase in cortical area fraction from baseline than Vivarium Control mice.

3.3. SF and HLU mice had lower femur trabecular bone volume than their respective controls. HLU mice had lower epiphyseal trabecular bone volume than SF mice

In the distal femur epiphysis, SF mice had significantly lower bone volume fraction (BV/TV) than Habitat Control mice (Fig. 2A). Similarly, HLU mice had significantly lower BV/TV than HLU Control mice. Both HLU Control and HLU groups had significantly lower BV/TV than Baseline, Vivarium Control, Habitat Control, and SF mice. HLU mice had the greatest decrease from baseline for epiphyseal BV/TV (Fig. 2B). Compared to HLU mice, SF mice had less bone loss (p < 0.05). In the distal femur metaphysis, SF mice had significantly lower BV/TV than Habitat Control mice (Fig. 2C). As it was in the epiphysis, both HLU Control and HLU groups had significantly lower BV/TV than Baseline, Vivarium Control, Habitat Control, and SF mice. SF mice had a significantly greater loss of BV/TV from baseline than Habitat Control mice (Fig. 2D).

3.4. SF and HLU mice had lower femur bone strength than their respective controls

Femurs from SF mice had significantly lower ultimate load, ultimate stress, and stiffness that Vivarium Control mice (Fig. 3A–C). Femurs from Vivarium Control mice had significantly greater ultimate load, ultimate stress, stiffness, and Young's modulus than Baseline mice (Fig. 3A–D). HLU mice had significantly lower ultimate load, ultimate stress, stiffness, and Young's modulus than HLU Control and SF mice.

Fig. 3.

Fig. 3

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Left femur mechanical properties as measured by three-point bending to failure (mean ± standard deviation). Unloading from spaceflight or hindlimb unloading (HLU) resulted in decreased femur ultimate load, ultimate stress, and stiffness, compared to Vivarium Control and HLU Control mice, respectively. Groups that share individual data point symbol style had the same mouse cage style. Groups that do not share a letter were significantly different (p < 0.05).

3.5. SF and HLU mice had lower gastrocnemius and quadriceps mass than their respective controls

Gastrocnemius and quadriceps mass in SF mice was not different than in Baseline mice (Fig. 4A–B). Vivarium Control mice had significantly greater quadriceps mass than SF and Baseline mice and significantly greater gastrocnemius mass than Baseline mice. HLU mice had significantly lower gastrocnemius and quadriceps mass than HLU Control mice. Similar results were observed when muscle mass was normalized by body weight (Fig. 4C–D).

Fig. 4.

Fig. 4

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Left gastrocnemius mass and quadriceps mass (mean ± standard deviation). Unloading from spaceflight or hindlimb unloading (HLU) resulted in decreased gastrocnemius mass and quadriceps mass, compared to Vivarium Control and HLU Control mice, respectively. Groups that share individual data point symbol style had the same mouse cage style. Groups that do not share a letter were significantly different (p < 0.05).

3.6. There were no differences in markers of muscle metabolism between SF, HLU and their respective controls

There were no differences between SF, Vivarium Control, and Habitat Control for expression levels of candidate skeletal muscle genes that are affected by SF (Fig. S1). HLU Control mice had significantly greater Fbxo32, Mstn, Cacng1, Myh4, and Myh7 expression than HLU mice and significantly greater Fbxo32, Mstn, Cacng1, Col1a1, Myh4, Myh7, and Fndc5 expression than SF mice. Quadriceps phosphorylated 4-ebp1, a marker of anabolic protein synthesis, was not significantly different between any groups (Fig. S2). This result suggests that changes in muscle protein synthesis that led to the differences in muscle mass observed at the end of the study may have occurred at an earlier timepoint in the study.

Supplemental Fig. S1.

Supplemental Fig. S1

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Left gastrocnemius muscle gene expression as measured by RT-qPCR (mean ± standard deviation). Genes typically affected by spaceflight were not significantly affected after six weeks of spaceflight. Groups that share individual data point symbol style had the same mouse cage style. Groups that do not share a letter were significantly different (p < 0.05).

Supplemental Fig. S2.

Supplemental Fig. S2

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Left quadriceps p-4ebp1/total 4ebp1 measured by Western blot (mean ± standard deviation). Groups that share individual data point symbol style had the same mouse cage style. There were no significant group differences.

4. Discussion

Spaceflight research is very costly, and there are limited opportunities. In order to develop countermeasures to the catabolic effects of spaceflight, Earth-based models that reproduce the effects of microgravity on the musculoskeletal system are needed for high-throughput screening of mechanisms of bone and skeletal muscle loss. HLU is the most widely used model of microgravity in animal research, but it has not been validated for long-duration SF, using parallel studies with mice of the same age, strain, sex, and experimental duration. In this study, we performed parallel long-duration SF and HLU experiments on skeletally immature, 10-week-old female Balb/c mice. SF and HLU mice both had decreased body weight, femur cortical area, femur trabecular bone volume, femur bone strength, quadriceps mass, and gastrocnemius mass, compared to their respective controls. Thus, in skeletally immature female Balb/c mice, both long-duration SF and HLU induced catabolic effects on bones and skeletal muscle, with HLU resulting in more pronounced skeletal deterioration.

Surprisingly, HLU mice had greater magnitudes of cortical and trabecular bone loss than SF mice, suggesting that HLU may be even more detrimental to bone health than SF. In this study, there were three different cage styles used. Mice in the Vivarium Control, Habitat Control, and HLU Control only differed by the type of cage the mice were housed in. Despite having the same loading environment, mice from these groups had significant differences in every bone property and in skeletal muscle mass. Differences in cage style include number of mice per cage, enrichment, cage floor bedding, and cage floor material. Cage floor bedding can affect mouse paw sensitivity, particularly when exposed to interventions that cause inflammation (Moehring et al., 2016). Stiffer cage bedding can increase paw sensitivity to touch, possibly leading to a reduction in physical activity. Mice in the HLU and HLU Control groups were housed in wire bottom cages, with floors made of stiff metal wires that did not have any bedding material. This may explain why both groups had typically worse bone and muscle outcomes than Vivarium Control mice, which were housed in cages with standard bedding during the experiment. Cage density may also have contributed to differences between SF and HLU mice. Mice in the HLU and HLU Control groups were housed two per cage while the Vivarium Control, Habitat Control, and SF groups were housed five per cage. Cage density does not affect animal wellbeing in group housed mice (Horn et al., 2012), but it can affect mouse body weight in Balb/c mice (Laber et al., 2008).

Mice in the HLU and HLU Control groups had lower initial body weight than the Vivarium Control, Habitat Control, and SF groups. This difference in body weight may have contributed to HLU and HLU Control mice having lower femur cortical area, femur ultimate load, and femur BV/TV than the Vivarium Control, Habitat Control, and SF groups. However, the percent change from baseline of HLU Control mice was no different than Vivarium Control mice for femur cortical area and trabecular BV/TV. These data suggest that initial body weight differences do not account for the differences in the response to SF and HLU. HLU more negatively affected quadriceps mass and femur cortical area, cortical area fraction, and epiphyseal BV/TV than SF. Change in body weight from baseline for HLU and HLU Control mice was less than what is expected from the breeder's growth charts, suggesting the cage style used for HLU may also negatively affect body weight in growing mice.

We did not detect changes in skeletal muscle gene expression or markers of protein synthesis in SF or HLU mice, compared to their controls. The genes we measured (Fbxo32, Trim63, Mstn, Cacng1, Col1a1, Myog, Myh4, Myh7, and Fndc5) are genes involved in muscle atrophy, muscle wound healing, and changes in mechanical loading. All the genes that we measured have been previously shown to be increased during SF or simulated microgravity (Smith et al., 2020; Hayashi et al., 2023; Bodine et al., 2001; Okada et al., 2021; Di Filippo et al., 2024; Hanson et al., 2013). However, these genes are typically acutely affected by microgravity. We measured gene expression after 39.5 and 35 days of SF and HLU, respectively. During unloading, muscle metabolism gene expression changes over time (Hanson et al., 2013). At the time point we studied, these genes likely would have returned to control levels via homeostatic mechanisms (Hanson et al., 2013; Sanesi et al., 2023). HLU Control mice had differences in gene expression compared to Vivarium Control, Habitat Control, and SF mice for almost every gene we measured. This may be due to differences in cage floor style as mentioned above. Additionally, we did not remove food from the cages to induce fasting prior to sacrifice, adding noise in the data. Fasting affects expression of muscle metabolism genes and protein synthesis markers, including those examined in this study (Jia et al., 2019). We do not know if mice were in a fasting or fed state when muscles were collected.

There were some limitations with our study. Mice in Baseline, Vivarium Control, Habitat Control, and SF groups were housed at KSC and the ISS, while mice in the HLU and HLU Control groups were housed in the vivarium at Virginia Commonwealth University. Differences in housing practices and protocols, including environmental conditions, diet, and water source may have contributed to differences in bone and muscle properties when comparing SF mice to those in our HLU study. Longitudinal in vivo CT scans were taken only for HLU mice, not SF mice. Thus, measurements of bone volume changes from baseline in SF mice are less accurate than measurements from HLU mice. All mice returned to normal gravity and ambulation prior to sacrifice, and for the SF mice this period was extended (by almost two days) potentially triggering acute increases in bone formation. Previous work in mice found that two days is not enough time to induce detectable increases in bone volume (Gerbaix et al., 2017). We initially intended to have the HLU mice in the tail suspension apparatus for 42 days. However, due to increased tail inflammation, we removed the mice from suspension after 35 days.

Long-duration SF induces harmful catabolic effects on the musculoskeletal system. In skeletally immature Balb/c mice, both SF and HLU produced comparable musculoskeletal deterioration, supporting the use of HLU to approximate select catabolic effects associated with unloading during long-duration SF. Use of HLU to model the effects of SF will be important for exploring cellular and molecular mechanisms regulating bone and skeletal muscle loss and for high throughput testing of therapeutic countermeasures.

The following are the supplementary data related to this article.

CRediT authorship contribution statement

Michael A. Friedman: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Yasmina Zeineddine: Investigation. Olivier Tuyambaze: Investigation. Wesam Elhawabri: Investigation. Ahmed Al Shammary: Investigation. Louis Stodieck: Writing – review & editing. Virginia L. Ferguson: Writing – review & editing. Henry J. Donahue: Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work is supported by the Translational Research Institute for Space Health Postdoctoral Fellowship (NASA Cooperative Agreement NNX16AO69A), Center for the Advancement of Science in Space User Agreement UA-2019-888, and National Institutes of Health 3UM1TR004360-02S2. Statistical consultation was provided by the VCU Wright Regional Center for Clinical and Translational Science.

Footnotes

☆

This article is part of a Special issue entitled: ‘Bone response to spaceflight’ published in Bone Reports.

Data availability

All raw data generated from this study is deposited online at doi: 10.17632/psjgcmy45h.1.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All raw data generated from this study is deposited online at doi: 10.17632/psjgcmy45h.1.

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