Figure 6

Synergistic effect on fibrosis induction in muscle of wild-type mice by combined treatments. (A) Sirius red and hematoxylin and eosin (H&E) staining of wild-type (WT) tibialis anterior (TA) muscles subjected to a combination of cardiotoxin (CTX) injury (50 μl of 10^–5 M) and denervation or transforming growth factor beta 1 (TGFβ1) (50 ng in 50 μl phosphate-buffered saline (PBS)) injection (as described in the Methods section), respectively, compared to CTX injury alone. (B) Quantification of collagen content in muscle after each treatment. Data correspond to the mean ± SEM; n = 4 on each group. Non-parametric Mann–Whitney U test; *P <0.05 versus CTX injury. (C) Quantitative RT-PCR for collagen I, connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinases 1 (TIMP-1) and TGFβ1 after the different treatments (n = 4 on each group. Non-parametric Mann–Whitney U test; *P <0.05, compared to CTX injury). (D) Representative immunostaining for collagen I (green) and fibronectin (red) on sections of WT muscle subjected to the different fibrosis-inducing methods. (E-G) Analysis of long-term fibrosis in WT muscle at one month after injury. Data are compared to non-injured (NI) muscle of sham-operated WT mice. (E) Sirius red and H&E staining of CTX-injured, lacerated, CTX/denervation and CTX/TGFβ-injured muscles at one month after injury. (F) Quantification of collagen content one month after injury of WT muscle. Values represent mean ± SEM; n = 4 on each group. Non-parametric Mann–Whitney U test; *P <0.05 versus NI. (G) Ex vivo maximum isometric force (tetanic force) of TA muscle. Values as mean ± SEM; n = 4 on each group. Non-parametric Mann–Whitney U test; *P <0.05 versus NI. Scale bars = 50 μm.