Influence of Social Isolation During Prolonged Simulated Weightlessness by Hindlimb Unloading

Abstract

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.

Keywords: HPA axis; bone loss; hindlimb unloading; immune system; microgravity model; muscle atrophy; social isolation; spaceflight.

FIGURE 1

Experiment design and groups.

FIGURE 2

Side view of (A) single housing HU and (B) social housing HU cage design.

FIGURE 3

Body weights from day 0 to 30 of hindlimb unloading (HU) and corresponding normally loaded (NL) controls in singly and socially housed female C57BL/6NJ mice. ^∗NL social and HU social groups display statistically significant differences at indicated days at p < 0.05 using two-way repeated measures ANOVA followed by Tukey post hoc test. Values are expressed as means. For clarity, error bars are omitted. Refer to Supplementary Table S1 for a full list of mean and SD values. Sample sizes: Single NL = 4; Single HU = 6; Social NL = 12; and Social HU = 12.

FIGURE 4

Soleus weights normalized to day 30 body weights of C57BL/6NJ mice. Social envt: Social environment. ^∗Statistically significant at p < 0.05 using two-way ANOVA followed by Tukey post hoc test. NS: Not significant.

FIGURE 5

Microcomputed tomography (μCT) analysis of cancellous compartment of proximal tibiae from C57BL/6NJ mice. (A) % Bone Volume, % BV/TV; (B) Trabecular Thickness, Tb.Th; (C) Trabecular Spacing, Tb.Sp; and (D) Trabecular Number, Tb.N. Two-way ANOVA was performed. NS, not significant.

FIGURE 6

Microcomputed tomography analysis of cortical compartment of tibiae from C57BL/6NJ mice. (A) Marrow Area, Ma.Ar; (B) Cortical Area, Ct.Ar; (C) Cortical Thickness, Ct.Th; (D) Periosteal Perimeter, Ps.Pm; and (E) Endocortical Perimeter, Ec.Pm. Two-way ANOVA was performed. NS, not significant.

FIGURE 7

Left and right (combined) adrenal weights normalized to day 30 body weights of C57BL/6NJ mice. ^∗Statistically significant at p < 0.05 using two-way ANOVA followed by Tukey post hoc test. NS: Not significant. Sample sizes: Single NL = 5; Single HU = 6; Social NL = 12; and Social HU = 12.

FIGURE 8

Plasma corticosterone levels of C57BL/6NJ mice. Two-way ANOVA with loglinear variance model was applied since unequal variance was observed. NS: Not significant. Sample sizes: Single NL = 8; Single HU = 5; Social NL = 12; and Social HU = 11.

FIGURE 9

Spleen weights normalized to day 30 body weights of C57BL/6NJ mice. Two-way ANOVA was performed. NS: Not significant.

FIGURE 10

Percentages of white blood cell subpopulations in peripheral blood of C57BL/6NJ mice. (A) and (B) were analyzed by two-way ANOVA while (C) and (D) had unequal variances and were therefore analyzed by two-way ANOVA with loglinear variance model. NS: Not significant. ^∗Statistically significant at p < 0.05 using Tukey post hoc test. NS: Not significant. Sample sizes: Single NL = 8; Single HU = 5; Social NL = 12; and Social HU = 12.