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

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

Schematic drawing of custom-made bite force device set up.

<p>Schematic drawing of custom-made bite force device set up.</p

Mean ± SE values of postural parameters.

<p>Mean and standard error values of center of pressure (COP) path length (A), area (B), velocity (C), and length function of surface (LFS) (D) during control period (BDC-3), the first day (R0) and the second day of recovery period (R+1) for the different test conditions: dental intercuspidal position (ICP) (white bars) and mandibular rest position (RP) (black bars), eyes open and eyes closed. * and ** significant differences compared with BDC-3 value. (Respectively <i>p</i> < 0.05 and <i>p</i> < 0.01; ANOVA for repeated measures).</p

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

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

Maximal molar bite force before and after dry immersion.

<p>Maximal molar bite force before and after dry immersion.</p

Combined (left and right) changes in jaw and cervical muscles stiffness, frequency and relaxation time before, during and after dry immersion.

<p>Combined (left and right) changes in jaw and cervical muscles stiffness, frequency and relaxation time before, during and after dry immersion.</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 temperature, body weight, plasma volume, systolic and diastolic pressure, and heart rate before and after dry immersion.

<p>Body temperature, body weight, plasma volume, systolic and diastolic pressure, and heart rate before and after dry immersion.</p