Research Paper Volume 11, Issue 15 pp 5628—5645

Rejuvenation of brain, liver and muscle by simultaneous pharmacological modulation of two signaling determinants, that change in opposite directions with age

Figure 5. p16 levels are decreased in situ in muscle and brain by Alk5i+OT. Cryosections of injured and uninjured muscle (10 µm, each) and brain (25 µm) were assayed for the number of p16+ nuclei by immunofluorescence, using Hoechst to label all nuclei. p16 showed the predicted nuclear localization in these assays. (A) Representative images of p16+ cells in the polymorphic layer near the dentate gyrus in an old control brain and isotype-matched IgG non-specific immunofluorescence are shown. Arrows point to p16+ (red)/Hoechst+ (blue) nuclei in the stated region. (B) The number of p16+ cells in the polymorphic layer of the hippocampus was decreased by the Alk5i+OT treatment, ***old control & Alk5i+OT p=0.0003, scale bar=50 µm at 40x magnification, IgG scale bar=50 µm at 20x magnification. Young control (Yc) n=5, old control (Oc) n=8, Alk5i+OT (OA5iOT) n=8. (C) Representative images of p16+ nuclei outside of the injury site in the TA muscle of an old vehicle control mouse at 5 days post single CTX injection and isotype-matched IgG non- specific immunofluorescence, are shown; scale bar=50 µm at 20x magnification. (D) The number of p16+/Hoechst+ nuclei divided by the total number of nuclei (Hoechst+) per field-of-view at 20x magnification (frequency of p16+ nuclei) was quantified. The frequency of p16+ nuclei outside of injury sites is significantly greater in muscle of old vehicle-treated control mice, as compared to young control and old Alk5i+OT treated. N=5 for each cohort. Young control (Yc), old+Alk5i+OT (OA5iOT), old control (Oc) N.S. = P-value Yc &OA5iOT = 0.064, *** = P-value Yc & Oc = 0.011, **** = P-value OA5iOT & Oc = 0.000064. (E) Representative images of the sites of injury/regeneration of young and old TA muscle from control - HBSS treated mice; 10-micron sections desmin (green), p16 (red) immunofluorescence and isotype-matched IgG control non-specific immune-fluorescence, are shown. Scale bar is 50 micron at 40x magnification. Robust p16+ nuclei are observed in both young and old muscle, and many of these are in centrally-nucleated newly formed desminhigh myofibers. The frequency of p16+ centrally-nucleated myofibers was quantified (right). The relative number of these p16+ fibers were found to be nearly identical in young injured (n=5) and old injured (n=7) muscle. N.S. = P-value Old and Young = 0.7918.