Figure 2

Quantification of fibrosis in muscle of mdx mice. (A) Sirius red and H&E staining of mdx tibialis anterior (TA) (upper panels) and diaphragm (lower panels) muscles at different ages compared to adult wild-type (WT) muscle. ‘Young’ corresponds to muscles of three-month-old mice, ‘Adult’ to nine months and ‘Old’ to eighteen to twenty-four months of age. (B) and (C) Active transforming growth factor beta 1 (TGFβ1) protein quantification by ELISA in TA and diaphragm muscles of WT and mdx mice at the indicated ages, respectively. Data correspond to the mean ± SEM values; n = 4 for each group. Two-way analysis of variance with Tukey’s post hoc multiple comparison test. **P <0.01, ***P <0.001 versus age-matched WT. (D) and (E) Quantification of collagen content in TA and diaphragm muscles of WT and mdx mice at different ages. Values are mean ± SEM; n = 4 for each group. Two-way analysis of variance with Tukey’s post hoc multiple comparison test. **P <0.01, ***P <0.001 versus control WT values. (F) Relative expression of collagen I, connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinases 1(TIMP-1) and TGFβ1 mRNA by quantitative RT-PCR in mdx TA muscles at the indicated ages with respect to WT muscles (baseline set arbitrarily to 1). Values are mean ± SEM; n = 3 for each group. Two-way analysis of variance with Tukey’s post hoc multiple comparison test. *P <0.05, **P <0.01, ***P <0.001, versus age-matched WT. (G) Representative pictures of immunofluorescence staining for collagen I (green) and fibronectin (red) in young, adult and old mdx TA, compared to WT muscle. Scale bars = 50 μm.