Fig 1.
3D printed-cast immobilization procedure.
(A) Picture of the two halves of different boots seizes. (B) Hindlimb immobilization procedure on a mouse anesthetized with isoflurane. (C) Time schedule of experimental groups with corresponding color code. Mouse draw adapted from BioRender.com (2020) licensed to University of Padova.
Fig 2.
Comprehensive muscle atrophy following two weeks of unilateral immobilization.
(A-B) After two weeks of disuse, the immobilized leg did not display edema, and the skin appeared intact. (C) Comparison between the gastrocnemius muscle of the free leg (left) and the contralateral muscle of the immobilized leg (right). (D) Comparison between free, immobilized, control gastrocnemius wet weight. (E) Representative images of Hematoxylin and Eosin of free and (F) immobilized gastrocnemius muscles. Scale bar = 100μm. (G) Myofibers cross-sectional area analysis of free, immobilized, control gastrocnemius. (H) the relative fiber size distribution. (leg n = 8 per each condition, mean±SD). Statistical significance was calculated using paired two-tailed Student’s t test. Data are mean±SEM (A to G), mean±SD (H). ** p<0,01; *** p<0,001; **** p<0,0001.
Fig 3.
Comprehensive muscle weakening after 2 weeks of unilateral immobilization.
Force-frequency curves were performed in vivo on gastrocnemius muscles. (A-B) Absolute force and (C-D) maximal specific force generated during tetanic contraction of control, free leg and immobilized one. (leg n = 8 per each condition) Statistical significance was calculated using paired two-tailed Student’s t test. Data are mean ± SEM, ** p < 0,01; *** p< 0,001, ****p<0,0001.
Fig 4.
Effects of a two-week immobilization period on myosin fiber types of gastrocnemius muscle.
(A-C) Comparison between free, immobilized, and free-roaming wild type legs of cross-sectional area variations according to fiber type and (D-F) of fiber type composition of the muscle. (G) Detail of fiber type composition of a free leg gastrocnemius, (H) an immobilized leg gastrocnemius and (I) a free roaming wildtype gastrocnemius (type I blue, type IIA green, type IIX black, type IIB red, dystrophin grey). Scale bar = 100μm; leg n = 5; magnification = 40x. (J) Immunofluorescence representative picture of free leg gastrocnemius cryosection stained for NCAM (red) and DAPI (blue), magnification = 20x (n = 5). (K) Immunofluorescence representative picture of immobilized leg gastrocnemius cryosection stained for NCAM (red) and DAPI (blue), n = 5; magnification = 20x. (L) Quantification of NCAM positive fibers in gastrocnemius of free, immobilized and free roaming wt animals (n = 5). Statistical significance was calculated using two-tailed Student’s t test. Data are mean±SEM, * p<0,05 ** p<0,01.
Fig 5.
Effects of 1 week immobilization.
(A) Quantitative RT-PCR of NJM genes expression in immobilized, free, and free roaming wild type leg after one week of immobilization, TBP normalized. (B) Comparison between free, immobilized, and free-roaming wild type legs of gastrocnemius muscle wet weight, (C) cross-sectional area and (D) relative fiber size distribution, and (E) Quantitative RT-PCR of atrogenes expression, TBP normalized (n = 5). Statistical significance was calculated using two-tailed Student’s t test. Data are mean±SEM (A, B, C, E), mean±SD (D). * p<0,05 ** p<0,01 *** p<0,001.
Fig 6.
Gait is not impaired after 2 weeks of immobilization. Components of the stride: (A) swing, (B) brake, (C) propel, (D) stance and (E) overall stride. (F) Difference in length covered by stride. (G) Stride frequency. (H) Number of steps. (I) Area covered by the paw at peak stance (leg n = 8 per each condition). Statistical significance was calculated using paired two-tailed Student’s T-test. Data are mean±SEM.
Fig 7.
After 1 week of recovery mice are still atrophic.
(A) Comparison between free and immobilized gastrocnemius wet weight. (B) Fiber size distribution (n = 3, mean±SD) and (C) myofibers cross-sectional area analysis of free and immobilized legs (leg n = 3 per each condition). (D) Representative images of Hematoxylin and Eosin of free and (E) immobilized gastrocnemius muscles. Scale bar = 100μm; magnification = 20x. Statistical significance was calculated using two-tailed Student’s t test. Data are mean±SEM (A and C), mean±SD (B). ** p<0,01 *** p<0,001.
Fig 8.
After 3 weeks of re-loading mice recovered completely.
(A) Comparison between free and immobilized gastrocnemius wet weight. (B) Fiber size distribution (n = 8, mean±SD) and (C) myofibers cross-sectional area analysis of free and immobilized legs. (D) Absolute force-frequency in vivo on gastrocnemius muscles and (E) specific force generated tetanic contraction of free leg and immobilized one (leg n = 8 per each condition). (F) Representative images of Hematoxylin and Eosin of free and (G) immobilized gastrocnemius muscles. Scale bar = 100μm; magnification = 20x. Statistical significance was calculated using paired two-tailed Student’s t test. Data are mean±SEM (A, C, D, E), mean±SD (B).