Research Paper Volume 4, Issue 8 pp 553—566

Figure 4. Modulation of osteopontin alters muscle regeneration in mice in vivo. (A) Schematic representation of in vivo modulation of OPN in young and old mice upon muscle injury. TA muscles of young and old mice were injured with CTX. 24-30 hours later (and subsequently daily for total of 4 days) the injured muscles of young mice were injected with PBS (right leg) or recombinant OPN (left leg); while the injured muscle of old mice were injected in the same regiment with goat IgG (right leg) and anti-OPN antibody (nOPN) in the left leg. Muscles were dissected on day 5 and analyzed for success in regeneration in 10 μm cryosections. (B) Muscle sections were stained for Hematoxylin and Eosin (H&E) and immuno-stained for embryonic myosin heavy chain (eMyHC), to visualize and quantify newly regenerating myofibers; representative images are shown. (C) Bar graph show mean and standard deviation of the numbers of new myofibers that were quantified based on the H&E staining; n=3, p≤0.05. (D) RNA isolated from Tibialis Anterior (TA) muscle of young and old mice that were treated as depicted in Figure 4 A were analyzed for myogenic marker gene expression by qRT-PCR. Old control mice have decreased expression of the studied myogenic markers as compared to young control mice. In vivo neutralization of OPN in muscle of old mice significantly induced MyoD, Myogenin and eMyHC myogenic markers, making their expression similar to that of young mice, and accordingly, the over expression of OPN in young mice considerably decreased the levels of these myogenic markers. Levels of young control mice (treated with PBS) were set as 1 (n=3 ±SEM p**≤0.05).