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 effect 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 HU housing displayed expected musculoskeletal deficits versus housing matched, normally loaded (NL) controls. However, select immune and 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.

Spaceflight poses many challenges for humans. Ground-based analogs typically focus on single parameters of spaceflight and their associated acute effects. This study assesses the long-term transcriptional effects following single and combination spaceflight analog conditions using the mouse model: simulated microgravity via hindlimb unloading (HLU) and/or low-dose γ-ray irradiation (LDR) for 21 days, followed by 4 months of readaptation. Changes in gene expression and epigenetic modifications in brain samples during readaptation were analyzed by whole transcriptome shotgun sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). The results showed minimal gene expression and cytosine methylation alterations at 4 months readaptation within single treatment conditions of HLU or LDR. In contrast, following combined HLU+LDR, gene expression and promoter methylation analyses showed multiple altered pathways involved in neurogenesis and neuroplasticity, the regulation of neuropeptides, and cellular signaling. In brief, neurological readaptation following combined chronic LDR and HLU is a dynamic process that involves pathways that regulate neuronal function and structure and may lead to late onset neurological sequelae.

Spaceflight entails exposure to numerous environmental challenges with the potential to contribute to both musculoskeletal and vascular dysfunction. The purpose of this review is to describe current understanding of microgravity and radiation impacts on the mammalian skeleton and associated vasculature at the level of the whole organism. Recent experiments from spaceflight and ground-based models have provided fresh insights into how these environmental stresses influence mechanisms that are related to redox signaling, oxidative stress, and tissue dysfunction. Emerging mechanistic knowledge on cellular defenses to radiation and other environmental stressors, including microgravity, are useful for both screening and developing interventions against spaceflight-induced deficits in bone and vascular function.

Space radiation may pose a risk to skeletal health during subsequent aging. Irradiation acutely stimulates bone remodeling in mice, although the long-term influence of space radiation on bone-forming potential (osteoblastogenesis) and possible adaptive mechanisms are not well understood. We hypothesized that ionizing radiation impairs osteoblastogenesis in an ion-type specific manner, with low doses capable of modulating expression of redox-related genes. 16-weeks old, male, C57BL6/J mice were exposed to low linear-energy-transfer (LET) protons (150 MeV/n) or high-LET ⁵⁶Fe ions (600 MeV/n) using either low (5 or 10 cGy) or high (50 or 200 cGy) doses at NASA’s Space Radiation Lab. Five weeks or one year after irradiation, tissues were harvested and analyzed by microcomputed tomography for cancellous microarchitecture and cortical geometry. Marrow-derived, adherent cells were grown under osteoblastogenic culture conditions. Cell lysates were analyzed by RT-PCR during the proliferative or mineralizing phase of growth, and differentiation was analyzed by imaging mineralized nodules. As expected, a high dose (200 cGy), but not lower doses, of either ⁵⁶Fe or protons caused a loss of cancellous bone volume/total volume. Marrow cells produced mineralized nodules ex vivo regardless of radiation type or dose; ⁵⁶Fe (200 cGy) inhibited osteoblastogenesis by more than 90% (5 weeks and 1 year post-IR). After 5 weeks, irradiation (protons or ⁵⁶Fe) caused few changes in gene expression levels during osteoblastogenesis, although a high dose ⁵⁶Fe (200 cGy) increased Catalase and Gadd45. The addition of exogenous superoxide dismutase (SOD) protected marrow-derived osteoprogenitors from the damaging effects of exposure to low-LET (¹³⁷Cs γ) when irradiated in vitro, but had limited protective effects on high-LET ⁵⁶Fe-exposed cells. In sum, either protons or ⁵⁶Fe at a relatively high dose (200 cGy) caused persistent bone loss, whereas only high-LET ⁵⁶Fe increased redox-related gene expression, albeit to a limited extent, and inhibited osteoblastogenesis. Doses below 50 cGy did not elicit widespread responses in any parameter measured. We conclude that high-LET irradiation at 200 cGy impaired osteoblastogenesis and regulated steady-state gene expression of select redox-related genes during osteoblastogenesis, which may contribute to persistent bone loss.

Summary: With the development of transcriptomic technologies, we are able to quantify precise changes in gene expression profiles from astronauts and other organisms exposed to spaceflight. Members of NASA GeneLab and GeneLab-associated analysis working groups (AWGs) have developed a consensus pipeline for analyzing short-read RNA-sequencing data from spaceflight-associated experiments. The pipeline includes quality control, read trimming, mapping, and gene quantification steps, culminating in the detection of differentially expressed genes. This data analysis pipeline and the results of its execution using data submitted to GeneLab are now all publicly available through the GeneLab database. We present here the full details and rationale for the construction of this pipeline in order to promote transparency, reproducibility and reusability of pipeline data, to provide a template for data processing of future spaceflight-relevant datasets, and to encourage cross-analysis of data from other databases with the data available in GeneLab.