Figure 3. Effect of SMT C1100 on in vivo utrophin levels in the mdx mouse. (A) Two-fold increase in utrophin mRNA following daily oral administration of mdx mice with SMT C1100 (50 mg/kg/day) or vehicle only (PBS-Tween-20 (0.1%)/5% DMSO) from three weeks of age for four weeks. Results represent the mean 6 S.E from six mice per treatment group and are corrected for b-actin. *p = 0.019; (B) Utrophin protein levels in heart and diaphragm following treatment of mdx mice as described in (A) above. Blots were stained with anti-utrophin (MANCHO3; 1:100) and ECL HRP-conjugated anti-mouse antibody (GE Healthcare). Bands were quantified using Image J and arbitrary units represent utrophin levels corrected for equal loading by a-actinin immunostaining. Results represent a mean 6S.E from eight mice per SMT C1100 Reduces mdx Dystrophic Symptoms treatment group except for heart vehicle only which is based on n = 7. *p,0.01; #p,0.05; (C) Immunohistochemical staining of EDL and TA muscle sections (10 mm; 206 magnification) were prepared from mdx mice treated as in (A). Sections were stained with anti-utrophin polyclonal antibody URD40 (1:100) and fluorescein isothiocyanate- conjugated anti-rabbit secondary antibody (1:1000). doi:10.1371/journal.pone.0019189.g003 level of regeneration taking place in skeletal muscle in mdx mice dosed with SMT C1100. This is demonstrated by a significant reduction in the numbers of fibres with centrally localized nuclei, as fibres with peripheral nuclei are thought to be more mature in development and therefore to have been a component of the muscle for longer (Fig. 4C). Significant reduction in centrally nucleated fibres is seen in the skeletal muscles examined including the diaphragm; a more severely affected mdx muscle which better mimics the more severe pathology of a DMD patient. As the cycle of fibre degeneration and regeneration is being slowed by continued utrophin expression in SMT C1100-dosed mdx then the cytoplasmic signals to engage in muscle repair responses such as inflammation and fibrosis are reduced. In normal muscle this inflammatory protection response is dampened down as the proliferation of resident satellite cells fuse and reconnects the broken fibres. However, with the constant degeneration, these protection signals are not switched off, resulting in the continued influx of inflammatory cells and fibroblasts, leading to an increasing cascade of further fibre damage and loss of muscle ‘‘space’’ by fibrotic plaques. Treatment with SMT C1100 significantly reduces this damage by virtue of the reduced fibre regeneration. Blinded analysis by a board- certified veterinary pathologist of muscle sections from mdx mice dosed with either vehicle or SMTC1100 demonstrated a significant reduction in both inflammation and fibrosis. Whole muscle sections were rated with a pathology score on a scale from normal (0) – mild (1) – moderate (2) – severe (3). Pooled averages of total scoring from the TA, EDL and soleus are shown (Fig. 5A). A qualitative example of the extent of inflammation from a SMT C1100-dosed EDL or vehicle dosed EDL (Fig. 5B) is shown where the SMT C1100 section was scored as mild (occasional mononuclear inflammatory cells in the inter-bundle connective tissue with focal aggregations of mononuclear inflammatory cells) and the mdx as moderate (multiple foci of mononuclear inflammatory cell infiltration in the inter-bundle connective tissue; occasional mononuclear inflammatory cells between individual muscle fibres). This data confirms the concept of reduced fibre damage due to utrophin localization leading to reduced inflam- mation and fibrosis. Benefits of daily dosing of forced exercise mdx mice with SMT C1100 A forced exercise regime of chronic exercise was used as a strategy to worsen the murine pathology [14,21]. Five week old mdx mice underwent forced treadmill exercise twice a week and the effects of daily SMT C1100 treatment under this exercise regime were then evaluated. This exercise protocol significantly worsens in vivo parameters readily evaluated by non-invasive approaches, such as fore limb grip and endurance tests. In particular, the exercise protocol induced the typical decrease of fore limb force in vivo over time; a reduction which is seldom observed in sedentary mdx. SMT C1100-treated mdx showed a significant protection against exercise-induced fore limb weakness, as demonstrated by both the maximal strength achieved and the increase in strength after four weeks of dosing (Fig. 6A, B). After four weeks of dosing both PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e19189