Figure 4

Induction of fibrosis after chemical and surgical muscle damage in young mdx mice. (A) Sirius red and hematoxylin and eosin (H&E) staining of tibialis anterior (TA) muscles of young (three-month-old) mdx mice two weeks after cardiotoxin (CTX)-injury (50 μl of 10^–5 M), denervation (DEN) and laceration (LAC), compared to non-injured (NI) muscle of sham-operated mdx mice. (B) Sirius red and H&E staining of young mdx TA muscle analyzed after two sequential treatments with recombinant transforming growth factor beta 1 (TGFβ1) (50 ng in 50 μl phosphate-buffered saline (PBS)) spaced seven days apart. (C) Biochemical quantification of collagen protein content in mdx TA, two weeks after different treatments relative to uninjured mdx control. Values represent mean ± SEM; n = 4 on each group. Non-parametric Mann–Whitney U test; *P <0.05 versus NI. (D) Quantitative RT-PCR of collagen I, connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinases 1(TIMP-1) and TGFβ1 mRNA expression in mdx muscle two weeks after different injuries versus control mdx mice. Values represent mean ± SEM; n = 4 on each group. Non-parametric Mann–Whitney U test; *P <0.05 versus NI. (E) Representative immunostaining for collagen I (green) and fibronectin (red) on sections of young mdx TA muscles two weeks after injury relative to control. (F) Sirius Red and H&E staining of mdx TA muscle two months after CTX injury, DEN, LAC or injection of TGFβ in three-month-old mdx muscle compared to NI mdx control muscle. (G) Quantification of collagen content in TA muscle of young mdx mice two months after different treatments, as described above. Values represent mean ± SEM; n = 4 on each group. Non-parametric Mann–Whitney U test; *P <0.05 versus NI. (H) Ex vivo maximum isometric force (tetanic force) of TA muscle of young mdx mice two months after treatments. Values as mean ± SEM; n = 4 to 5 on each group. Non-parametric Mann–Whitney U test; *P <0.05; **P <0.01 versus NI. Scale bars = 50 μm.