Table_4_Influence of Social Isolation During Prolonged Simulated Weightlessness by Hindlimb Unloading.DOCX

The hindlimb unloading (HU) model has been used extensively to simulate the cephalad fluid shift and musculoskeletal disuse observed in spaceflight with its application expanding to study immune, cardiovascular and central nervous system responses, among others. Most HU studies are performed with singly housed animals, although social isolation also can substantially impact behavior and physiology, and therefore may confound HU experimental results. Other HU variants that allow for paired housing have been developed although no systematic assessment has been made to understand the effects of social isolation on HU outcomes. Hence, we aimed to determine the contribution of social isolation to tissue responses to HU. To accomplish this, we developed a refinement to the traditional NASA Ames single housing HU system to accommodate social housing in pairs, retaining desirable features of the original design. We conducted a 30-day HU experiment with adult, female mice that were either singly or socially housed. HU animals in both single and social housing displayed expected musculoskeletal deficits versus housing matched, normally loaded (NL) controls. However, select immune and hypothalamic-pituitary-adrenal (HPA) axis responses were differentially impacted by the HU social environment relative to matched NL controls. HU led to a reduction in % CD4+ T cells in singly housed, but not in socially housed mice. Unexpectedly, HU increased adrenal gland mass in socially housed but not singly housed mice, while social isolation increased adrenal gland mass in NL controls. HU also led to elevated plasma corticosterone levels at day 30 in both singly and socially housed mice. Thus, musculoskeletal responses to simulated weightlessness are similar regardless of social environment with a few differences in adrenal and immune responses. Our findings show that combined stressors can mask, not only exacerbate, select responses to HU. These findings further expand the utility of the HU model for studying possible combined effects of spaceflight stressors.后肢悬吊模型（hindlimb unloading, HU）已被广泛用于模拟航天飞行中观察到的头向体液转移与肌肉骨骼废用状态，其应用范围现已拓展至免疫、心血管及中枢神经系统应答等领域的研究。绝大多数HU实验均采用单笼饲养的动物开展，但社会隔离也会显著影响动物的行为与生理状态，进而可能混淆HU实验的结果。目前虽已开发出支持成对饲养的HU改良模型，但尚未有系统评估研究阐明社会隔离对HU实验结局的影响。因此，本研究旨在明确社会隔离对HU模型下组织应答的贡献。为此，我们对传统NASA Ames单笼饲养HU系统进行了改良，使其可支持成对社交饲养，同时保留原设计的优良特性。我们采用单笼饲养或社交饲养的成年雌性小鼠开展了为期30天的HU实验。与饲养条件匹配的正常负重（normal loaded, NL）对照组相比，单笼与社交饲养的HU组小鼠均出现了预期的肌肉骨骼功能缺陷。然而，相较于匹配的NL对照组，部分免疫与下丘脑-垂体-肾上腺（hypothalamic-pituitary-adrenal, HPA）轴应答会因HU饲养的社交环境不同而产生差异。HU会使单笼饲养小鼠的CD4+ T细胞占比降低，但对社交饲养小鼠无此影响。出乎意料的是，HU会使社交饲养小鼠的肾上腺重量增加，而单笼饲养小鼠无此变化；同时在NL对照组中，社会隔离会提升肾上腺重量。此外，单笼与社交饲养的HU组小鼠在第30天时的血浆皮质酮水平均有所升高。综上，无论社交环境如何，小鼠对模拟失重的肌肉骨骼应答均无显著差异，仅肾上腺与免疫应答存在少量差异。本研究结果表明，多重应激源不仅会加重，还会掩盖部分HU相关应答。上述发现进一步拓展了HU模型在研究航天飞行应激源联合效应方面的应用价值。