Fig. 2

miR-27b improves perfusion in murine model of critical limb ischemia. Hind limb ischemia (HLI) was induced in FVB mice by surgical ligation of the femoral arteries on day 0. Laser Doppler scans (LDS) have been performed for each animal prior to surgery (day −1), after ligation (day 0) to confirm the onset of ischemia, and on days 5 and 7, to assess recovery. On day 0, mice were randomly assigned to groups (n = 5) and injected three times with negative control (RNAi) or miR-27b mimic (10 μg/injection/day, days 0–2; NC and 27b, respectively). Tissues were harvested and processed on day 14. (a) At endpoint (day 14) calf muscles were harvested and snap-frozen for analysis. Five-μm crosswise sections were stained for the endothelial marker, CD31 (red), to assess microvessel density (MVD). Representative photomicrographs are shown. (b, c) CD31-positive vascular area was measured using Elements software (Nikon) on the images of at least nine fields from three independent sections (×20 objective). The data are presented as box plots with whiskers. P values were calculated using Tukey’s Multiple Comparisons test (b) Note increased vascular area in ischemic tissue upon miR-27 treatment (27b) compared to control (NC). (c). To correct for the baseline MVD in each animal, the ratio between ischemic (HLI) and non-ischemic (control) limb was calculated for miR-27b and control-treated animals. P value was calculated using one-sample t test. (d) The representative LDS images for days 0 and 7. Color scale for perfusion range is shown, with blue indicating impeded perfusion. Ischemic legs are indicated by arrows. (e) The perfusion was quantified and normalized against control (intact) limb; mean values and S.E.M. were calculated for each time point. P values were calculated by the Two-way RM ANOVA and Bonferroni posttest. Note progressive improvement of blood flow in ischemic limbs in miR-27b group (27b) compared to control treated group (NC)