MRI scans were acquired along the length of the right tibia (for triceps surae muscles) and femur (for quadriceps muscles) using a magnetic resonance imaging (MRI; 1.5 T Signa HDx, General Electric, Milwaukee, WI, USA). Participants laid on the MRI table on their back. The ankle was held at 90° by a custom wooden square to ensure the same position between participants and test sessions. A specific antenna for the acquisition of muscle cross-sectional areas (CSA) was installed (covering 45 cm). The femoral quadriceps and triceps surae muscles were scanned using a VIBE (Volume interpolated GRE) image sequence (Matrix 240 × 240; TR 6.3 ms; TE 3.0 ms, cutting thickness 2.6 mm, gap = 0). An axial scan was performed perpendicular to the thigh, from the femoro-tibial joint to the iliac crest for the quadriceps muscles, and from the calcaneus to the femoro-tibial joint for triceps surae muscles. Due to the length of the antenna, the quadriceps scan had to be performed with two sequences for some tall participants (n = 14). In this case, external markers (oil capsules) were placed on the participant’s leg using adhesive tissue to be able to adjust and overlap the different acquisition sequences in post treatment.

The accuracy of using serial ACSA scans from MRI for the measurement of muscle volume has been reported previously [19, 33]. The anatomical cross-sectional area (ACSA) was measured by an image processing software (Simpleware ScanIP, Synopsys Inc., Exeter, United Kingdom) throughout the excursion of each of the individual muscles composing the two muscle groups of interest: triceps surae and quadriceps. The distal and proximal insertions of each of the muscles analyzed were identified before starting the analysis. For each of the seven individual muscles of interest (quadriceps muscles (VL, VI, VM, RF) and triceps surae muscles (GL. GM, soleus)), segmentation was performed every eight slices (18.2 mm). In the first of this analysis, the ACSA of each muscle was tracked manually and measured digitally, and this was done for the three test sessions of a same participant by the same investigator. Furthermore, a grayscale thresholding of the MRI image was used to identify the aponeurotic limit. In a second step, visible fat and connective tissue were excluded from the measurement to only take into account the contractile part of the muscle volume [34]. To separate the non-contractile tissue, a region of interest (ROI) containing muscle and subcutaneous fat was made. Then, a histogram of signal intensity within the ROI was produced. To separate contractile and non-contractile tissues with minimal investigator bias, thresholding was performed by the Maximum Entropy method, a reliable histogram shape-based technique used in medical imaging analysis [35]. Pixel values above the threshold were considered to be non-contractile tissue [18]. Finally, this area, comprising fat and connective tissue, was then subtracted from the total muscle area, in order to measure only the CSA of the contractile components.

To be able to compare the ACSA of each participant according to their muscle length, the position of the ACSA was expressed according to the relative position of the ACSA in relation to the total length (L) of the muscle of interest. The position corresponding to 0% muscle length represented distal insertion for the quadriceps muscles, and proximal insertion for the triceps surae muscles with 100% representing the proximal and distal insertions for the quadriceps and triceps surae muscles respectively [22, 36]. To compare the evolution profiles of the ACSA with age and training, the ACSA values obtained were normalized to the maximum ACSA (ACSAmax, cm2) value reached for each of the muscles analyzed. The evolution of the ACSA of each of the seven muscles of interest was then expressed as a function of the relative length of the muscle and a polynomial regression curve was fitted to the data.

The volume of each muscle was estimated by integrating the regression equation (function of ACSAmax) over the length of the muscle (L) using the formula:

The length of the muscle (L, cm) is the distance between the proximal and distal insertion of the considered muscle (corresponding to the number of MRI slices x slice thickness (2.6 mm)) [22].

The statistical analyses were performed with SigmaPlot 11.0 software (Systat Software, San Jose, USA).

Root mean square (RMS) differences were used to assess reproducibility from the two pre-training data sets (4 weeks and just before the beginning of training) on regression curves. Intraclass correlation (ICC) was calculated for muscle volume comparison from both measurement sets.

Two-way analysis of variance (ANOVA) (Holm–Sidak multiple comparisons post hoc testing) with distance (every 25% of the relative length of the muscle) and age (between group young vs. old) factors, was used to determine differences on the mean value of ACSA in the atrophy in individual muscles. Similarly, the effect of training on the mean value of ACSA within group (pre vs. post) every 25% of the relative length of the muscle was analyzed using the same two-way ANOVA.

After testing the normality distribution using a Shapiro-Wilk test, the comparison of the volume between pre and post-test, was tested by a Paired t-test. Values were considered significant at an alpha level of P < 0.05. The effect of training between the three different groups and between quadriceps and TS muscles within the same group (Y55, O55, O80) were analyzed using two-way ANOVA, with Holm–Sidak multiple comparisons post hoc testing. Data are presented as means ± SD.

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