Fig 1.
Micro-CT evaluation of the hind limb muscle mass and definition of the index of muscle mass (IMM).
(A) Mice were placed prone on the micro-CT bed leaving the hind limbs in a natural position (foot and tibia angle mean ± SD: 41° ± 6, n = 30). (B) Scanned raw images were reconstructed in 3D and analyzed using Micro View analysis software. Scale bar, 2 mm. (C) For the evaluation of the muscle mass, the following parameters from 3D reconstructed images were measured: the distance from the upper extremity of the tibia (ut) to the medial malleolus (m), defined as the length of the tibia (L), and the perpendicular distance from the half-length of the tibia to the external margin of the hind limb muscle, referred to as the thickness of the muscle (T). These measurements were assessed on the plane in which the patella (p) and the upper extremity of tibia (ut) were clearly visualized. Scale bar, 2 mm.
Table 1.
CV of repeated measurements of IMM in the same mouse at three different times.
Fig 2.
SOD1G93A mice have a lower hind limb muscle mass than Ntg controls already at a presymptomatic stage of the disease.
(A-B) Grip strength as latency to fall (sec) and body weights (g) in Ntg and SOD1G93A mice are reported from 11 to 22 weeks of age (n = 10 per group). Data are expressed as mean ± SEM. *, p < 0.05 by two-way ANOVA for repeated measures (the interaction between genotype and time was significant in A and B with p < 0.0001), Bonferroni’s post-hoc test. (C-D) The weight of TA (C) and Gastrocnemius (GC) (D) was measured in Ntg and SOD1G93A mice at 12, 14, 17 and 20 weeks of age (n = 5 for each group). The weight of TA and GC was lower in SOD1G93A mice than age-matched Ntg controls already at a presymptomatic stage of the disease (12 weeks of age). Muscle weight was expressed in mg. Decrease in % was reported with respect to Ntg mice at 12 weeks of age. Data (mean ± SEM) were significantly different (p < 0.05), by one-way ANOVA, Newman-Keuls post-hoc test: *, versus Ntg; #, versus 12-week old SOD1G93A; °, versus 14-week old SOD1G93A;§, versus 17-week old SOD1G93A mice.
Fig 3.
IMM correlates with hind limb muscle weight in SOD1G93A mice as disease progresses.
(A) IMM was evaluated in Ntg and longitudinally in SOD1G93A mice at 12, 14, 17 and 20 weeks of age (n = 5 for each group). Decrease in % was reported respect to Ntg mice at 12 weeks of age. Data, expressed as mean ± SEM, were statistically significant different (p < 0.05), by one-way ANOVA, Newman-Keuls post-hoc test: *, versus Ntg; #, versus 12-week old SOD1G93A mice. (B-C) A correlation analysis was done with the mean values of the IMM and of the muscle weight of TA (B) and Gastrocnemius (GC) (C) for the different experimental groups. The analysis confirmed a significant (p < 0.008 for TA, p < 0.005 for GC) direct correlation between the two type of measurements with r2 = 0.9293 and r2 = 0.9502 for TA and GC respectively. Data are expressed as mean ± SEM.
Fig 4.
C26-bearing mice display hind limb muscle mass loss at 12 days from tumor injection.
(A-B) Grip strength (N) and body weight loss (%) are shown for C26-bearing mice and PBS-injected ones (n = 3 and n = 4, respectively). Data are expressed as mean ± SEM. *, p < 0.05 by two-way ANOVA for repeated measures (the interaction between treatment and time was significant in A with p = 0.0002 and in B with p < 0.0001), Bonferroni’s post-hoc test. (C) Muscle mass loss evaluated by micro-CT according to IMM definition in C26-bearing mice and PBS-injected ones (n = 11 and n = 4, respectively). Data are expressed as mean ± SEM. *, p < 0.05 by two-way ANOVA for repeated measures, Bonferroni’s post-hoc test. (D-E) Muscle weights of TA (D) and GC (E) of C26-bearing mice and PBS-injected ones are reported 12 days after tumor inoculation (n = 4). Data are expressed as mean ± SEM. * p < 0.05 by Student’s t-test, versus PBS. Both muscle weights are expressed in mg. (F) A similar result was obtained by evaluating the IMM as an indicator of muscle mass in the same set of mice (n = 4). Data are expressed as mean ± SEM, and were statistically significant different (p < 0.05) by the Student’s t-test.