Body weight gain, lymphoid organ normalized weight and spleen cellularity in control (C), restrained (R) and hindlimb unloaded (HU) mice at the end of the experiments.

<p>C  =  Control, R  =  Restrained, HU  =  Hindlimb Unloaded.</p><p>* <i>p</i> = 0.032 <i>versus</i> R, ^¤<i>p</i> = 0.002 <i>versus</i> C.</p><p>Body weight gain was calculated with the formula [body weight on day 21 - body weight on day 0 of the treatment]. Normalized weights were calculated with the ratio [organ weight/body weight] for each mouse. The number of splenic nucleated cells was calculated after red blood cell lysis with NH₄Cl. Each group was compared to the others. <i>n</i> = 5 mice per group. Differences were found to be statistically significant using an ANOVA and Tukey <i>post-hoc</i> test. HU mice did not gain weight (<i>p</i> = 0.032 <i>versus</i> R, <i>p</i> = 0.002 <i>versus</i> C), while the other groups grew by approximately 7% (R) to 11% (C) in comparison to their initial weight. No significant difference was found between the three experimental groups for lymphoid organ normalized weights. The number of nucleated cells was reduced by 19% and 25%, respectively, in the R and HU mice in comparison to C mice, although these differences were not significant (<i>versus</i> R: <i>p</i> = 0.479; <i>versus</i> HU: <i>p</i> = 0.273). Data are mean values ± SEM.</p

Cytokines secreted by splenic lymphocytes stimulated with LPS.

<p>Splenocytes were incubated with 5 μg/ml of LPS for 72 h. Cytokine concentrations in the supernatants were determined by flow cytometry using a Flowcytomix kit. Each group, hindlimb unloaded (HU), restrained (R) and control (C), was compared to the others. <i>n</i> = 5 mice per group. No significant difference was found between the three experimental groups using ANOVA and Tukey <i>post-hoc</i> test. Cytokines whose concentrations were below the detection threshold of the kit are not indicated.</p

Serum corticosterone concentration in control (C), restrained (R) and hindlimb unloaded (HU) mice.

<p>Trunk blood was collected immediately after the sacrifice. Corticosterone concentration was measured using an ELISA kit with a detection threshold of 16.9/ml. Each experimental group was compared to the others. <i>n</i> = 4, 6 and 4 mice for C, R and HU groups, respectively. No significant difference was found using an ANOVA and Tukey <i>post-hoc</i> test.</p

Variations of lymphocyte populations 72

<p>C  =  Control, R  =  Restrained, HU  =  Hindlimb Unloaded.</p><p>* <i>p</i>  =  0.025 <i>versus</i> C. ^¤<i>p</i>  =  0.081 <i>versus</i> C.</p><p>After 72 hours of culture, cells were incubated with fluorescent antibodies before identification by flow cytometry. Specific subpopulations of lymphocytes were determined within the lymphocyte gate. The formula [(% population stimulated - % population unstimulated)/% population unstimulated] was used to calculate the percentage of variation after stimulation with mitogens. Each group was compared to the others. <i>n</i>  =  5 mice per group. Statistically significant differences were found using an ANOVA and Tukey <i>post-hoc</i> tests. After LPS stimulation, the number of total lymphocytes was increased by only 16.9% in the hindlimb unloaded (HU) group compared to 41.6% in control (C) mice, corresponding to a reduction of 60% that was significantly different (<i>p</i>  =  0.025 <i>versus</i> C). Data are mean values ± SEM.</p

Phenotypes of splenic lymphocytes from control (C), restrained (R) and hindlimb unloaded (HU) mice after 72 h of incubation without mitogen.

<p>After incubation with fluorescent antibodies, cell populations were identified by flow cytometry. Lymphocytes were first gated according to their size and granularity. Populations of interest were then expressed as percentages of the lymphocyte population. Each experimental group was compared to the others. <i>n</i> = 5 mice per group. No significant difference was found using an ANOVA and Tukey <i>post-hoc</i> test.</p

Detection of lymphocyte division after 72<i>in vitro</i> stimulation with or without mitogen.

<p>Splenocytes were labeled with CFSE and cultured for 72(LPS or ConA). For each mouse, dividing lymphocytes were determined as in the three examples presented here. (<b>A</b>) Cells were labeled with fluorescent antibodies and analyzed by flow cytometry. They were sorted with FSC/SSC profiles, separating viable lymphocytes (black arrow) from dead cells and cellular debris. (<b>B</b>) Among the viable lymphocyte gate, each subpopulation was selected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092664#pone-0092664-g003" target="_blank">Figure 3</a> and assessed for fluorescent division peaks. Generation 0 (black arrow) and each of the following generations (pink area) were used by the software to calculate the number of dividing cells.</p

Absolute numbers of lymphocytes and lymphocytes subsets in the spleen (x10⁶).

<p>C  =  Control, R  =  Restrained, HU  =  Hindlimb Unloaded.</p><p>* <i>p</i> = 0.039 <i>versus</i> C; ^¤<i>p</i> = 0.011 <i>versus</i> C.</p><p>Absolute numbers for each cell population was calculated using the number of splenic nucleated cells and the percentages obtained by flow cytometry. Each group was compared to the others. <i>n</i> = 4, 5 and 4 mice for C, R and HU groups, respectively. Differences were found to be statistically significant using an ANOVA and Tukey <i>post-hoc</i> test. The number of total lymphocytes was reduced by 25% and 33%, respectively, in R and HU mice in comparison to C mice. There was no significant difference for T cells and their subpopulations for the three groups. The number of B cells was significantly decreased by 44% (<i>p</i> = 0.039) and 59% (<i>p</i> = 0.011) in R and HU mice, respectively. Data are mean values ± SEM.</p

Body weight evolution in control (C), restrained (R) and hindlimb unloaded (HU) mice.

<p>Mice of each group were weighed daily for the first four days and then every two days. Every seven days, cages were cleaned and feeders and bottles were refilled with food and water. Weights are presented with SEM for each condition of housing. Each experimental group was compared to the others. <i>n</i> = 5 mice per group. Differences were found to be statistically significant using ANOVA and a Tukey <i>post-hoc</i> test. * <i>p</i> = 0.035 HU <i>versus</i> C by day 19; ^T<i>p</i> = 0.068 HU <i>versus</i> C by day 21.</p

Representative flow cytometry density plots from splenic lymphocyte populations.

<p>Splenocytes were isolated from mice and labeled as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092664#s2" target="_blank">Methods</a> section. For each mouse, the lymphocyte populations were determined as in the two examples presented here. (<b>A</b>) Splenocyte analysis by flow cytometry using FlowJo software and sorted with FSC/SSC profiles, separating viable lymphocytes (black arrow) from the other cells. (<b>B</b>) Among the lymphocyte population, CD19⁺ B cells (black arrow) were selected in the FSC/CD19 window and CD3⁺ T cells (white arrow) were selected in the FSC/CD3 window. (<b>C</b>) Among the lymphocyte population, CD3⁺CD8⁺ T cells (black arrow) were selected in CD3/CD8 profiles and CD3⁺CD4⁺ T cells (white arrow) were selected in CD3/CD4 profiles.</p

Cytokines secreted by splenic lymphocytes stimulated with ConA.

<p>Splenocytes were incubated with 5 μg/ml of ConA for 72 h. Cytokine concentrations in the supernatants were determined by flow cytometry using a Flowcytomix kit. Each group, hindlimb unloaded (HU), restrained (R) and control (C), was compared to the others. <i>n</i> = 5 mice per group. No significant difference was found between the three experimental groups using ANOVA and Tukey <i>post-hoc</i> test. Cytokines whose concentrations were below the detection threshold of the kit are not indicated.</p