Elsevier

Acta Astronautica

Volume 92, Issue 1, November 2013, Pages 89-96
Acta Astronautica

Increasing the number of unloading/reambulation cycles does not adversely impact body composition and lumbar bone mineral density but reduces tissue sensitivity

https://doi.org/10.1016/j.actaastro.2012.04.003Get rights and content

Abstract

A single exposure to hindlimb unloading leads to changes in body mass, body composition and bone, but the consequences of multiple exposures are not yet understood. Within a 18 week period, adult C57BL/6 male mice were exposed to 1 (1x-HLU), 2 (2x-HLU) or 3 (3x-HLU) cycles of 2 weeks of hindlimb unloading (HLU) followed by 4 weeks of reambulation (RA), or served as ambulatory age-matched controls. In vivo μCT longitudinally tracked changes in abdominal adipose and lean tissues, lumbar vertebral apparent volumetric bone mineral density (vBMD) and upper hindlimb muscle cross-sectional area before and after the final HLU and RA cycle. During the final HLU cycle, significant decreases in total adipose tissue and vertebral vBMD in the three experimental groups occurred such that there were no significant between-group differences at the beginning of the final RA cycle. However, the magnitude of the HLU induced losses diminished in mice undergoing their 2nd or 3rd HLU cycle. Irrespective of the number of HLU/RA cycles, total adipose tissue and vertebral vBMD recovered and were no different from age-matched controls after the final RA period. In contrast, upper hindlimb muscle cross-sectional area was significantly lower than controls in all unloaded groups after the final RA period. These results suggest that tissues in the abdominal region are more resilient to multiple bouts of unloading and more amenable to recovery during reambulation than the peripheral musculoskeletal system.

Highlights

► Unloading significantly reduced lumbar vBMD and adipose tissue. ► Consecutive unloading cycles diminished the rate loss of lumbar vBMD and adipose tissue upon subsequent loading cycles. ► Lumbar vBMD and adipose tissue recovered post-reambulation irrespective of loss. ► Lean tissue volume was less affected by multiple unloading cycles.

Introduction

Exposure to microgravity can profoundly alter body mass, body composition, and function of the musculoskeletal system [1], [2]. While body and muscle mass may return to normal values within several months upon return weight-bearing [1], weight-bearing bones may not recover even years after returning to normal ambulatory activities [3]. Because of the limited number of space-missions and the invasive nature of many assays, studies have turned to the well-established rodent hindlimb unloading model (HLU) to investigate the mechanisms by which a single unloading period causes atrophy [4]. However, the impact of repeated exposures to microgravity on many physiologic systems, including the musculoskeleton and body composition, is largely unknown.

Decrease in body mass during spaceflight, bedrest, and HLU may occur via losses in adipose and/or lean tissue, and have been attributed to many factors such as reduced caloric intake, dehydration, as well as increased (e.g., during HLU) or decreased energy expenditure (e.g., during bedrest) [1], [5], [6], [7], [8]. During hindlimb unloading, body mass and serum leptin levels, linking energy metabolism to bone mass, can diminish even when caloric intake is adequate [9]. Importantly, after 28 days of HLU, the change in bone formation rates was significantly correlated with the change in serum leptin levels [9]. However, the loss of functional weightbearing also alters the mechanical milieu of bone marrow mesenchymal stem cells (MSCs), the pluripotent precursors to numerous cell types including osteoblasts and adipocytes [10], [11], [12], which may bias the differentiation of MSCs from an osteogenic to an adipogenic lineage. Whether unloading-induced changes in MSC differentiation result in increased abdominal adipose tissue is not well understood.

Further, abdominal adipose tissue is distributed as either subcutaneous adipose tissue (SAT) or visceral adipose tissue (VAT). While abdominal adipose tissue in general is associated with obesity-related health problems [13], VAT in particular is linked to increased metabolic risk [14], [15], [16]. In addition, VAT is lost preferentially over SAT during weight loss [17]. Little is known, however, whether the preferential loss of VAT also occurs in the mouse during hindlimb unloading.

Microgravity and HLU also reduce lean tissue mass and muscle size [1], associated with muscle atrophy in postural muscles containing primarily slow-twitch muscle fibers [18], [19]. Bone receives mechanical input both from muscle forces as well as from ground reaction forces during gravitational impact [20]. As ground reaction forces are removed during HLU, muscle activity provides the dominant source of mechanical cues [21]. In contrast, ambulatory activities upon release from HLU engage both muscle and impact with the ground, perhaps disassociating muscle and bone recovery. While muscle-bone relations during disuse have been investigated in the hindlimbs [21], [22], [23], the extent by which muscle forces maintain vertebral bone is much less understood. And even though bone loss throughout the skeleton environment is site-specific in microgravity [24], it is largely unknown whether the response of the spine to HLU and reambulation (RA) is similar to peripheral skeletal sites [25].

We recently demonstrated in C57BL/6 mice that overall, multiple exposures to hindlimb unloading were more detrimental than a single unloading cycle to both cortical and trabecular bone in the distal femur metaphysis [26]. Nevertheless, the magnitude of bone loss diminished during the 2nd and 3rd unloading cycle. Whether these 2 phenomena observed in the appendicular skeleton extend to lumbar vertebrae, abdominal lean tissue volume, muscle cross-sectional area, and adipose tissue is unknown. Here, using new data collected during the previous experiment [26], we hypothesized that because of bone's incomplete ability to recover during RA, multiple unloading cycles would be more detrimental than a single unloading cycle to lumbar apparent bone mineral density. We also hypothesized that previous exposures to unloading will attenuate the response of fat, muscle and bone to subsequent loading cycles. Further, based on associations between serum leptin and bone loss, we hypothesized a positive, linear relationship between bone loss and adipose tissue loss, as well as bone loss and muscle loss.

Section snippets

Experimental design

All procedures were approved by the Stony Brook University Animal Care and Use Committee. Forty-four young adult male, 16 week old C57BL/6 J (B6) mice (The Jackson Laboratory, Bar Harbor, ME) were weighed and randomly assigned to either age-matched controls (control, n=9) or to single (1x-HLU), double (2x-HLU), or triple (3x-HLU) unloading and reambulation (RA) cycles (n=11/group). Unloading was achieved by elevating the hindlimbs through tail suspension, simulating the effects of microgravity,

Body mass

At baseline, mice assigned to all groups had similar mean body mass (27.5±0.5 g). All mice remained healthy and active throughout the protocol. During the final unloading cycle, all unloaded groups lost body mass in comparison to controls, and animals undergoing their first unloading cycle lost significantly more (10±3%) than 3x-HLU (6+3%, p<0.001, Fig. 2). However, body mass recovered during the last reambulation cycle as all unloaded groups increased by a similar extent (3%, p<0.001) and there

Discussion

Deterioration of the musculoskeleton and changes in abdominal tissue composition have been reported previously for rodents exposed to hindlimb unloading but the effects of multiple exposures are unknown. We found that in male C57BL/6 mice, while the absolute tissue quantity reached after the final unloading cycle was similar regardless of the number of previous unloading cycles, previous exposure to unloading cycles attenuated the change in adipose, lean and bone tissue during the last

Acknowledgments

This research was kindly funded by NASA, a Kirchman T32 postdoctoral fellowship from the NIDDK, and NSERC. Technical support from Gunes Uzer, Surabhi Vijayaraghavan, Alyssa Tuthill, Andrea Trinward, and Svetlana Lublinsky was greatly appreciated.

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