Fig. 3

Histological assessments of the VL and soleus muscle in control diet (chow) and HFS diet group rats at 24-week-old and following 12 weeks of HFS diet exposure for the experimental male and female Sprague–Dawley rats. (A) Oil Red O staining for intramuscular lipids in VL and soleus muscle sections in male and female animals taken at 100× magnification. (B, C) raw values for % Oil Red O staining per cross-sectional mid-belly muscle area represented by box and whisker plots. (B) intramuscular fat in the VL was increased after 12-weeks in the HFS diet compared to the chow control animals for both male and females (C) intramuscular fat in the soleus only increased in the female HFS animals following the 12-week metabolic challenge. (D) Picrosirius Red staining for collagen in both VL and soleus muscle sections in male and female animals taken at 100× magnification. (E, F) raw values for % Picrosirius Red staining per cross-sectional muscle area represented by box and whisker plots. No statistically significant difference was found in % Picrosirius Red staining between groups for both the VL and soleus muscle. (G) Immunohistochemistry staining for CD68 + cells in VL and soleus muscle sections, imaged at 200× magnification. Yellow/red CD68 + stain overlapping with blue DAPI stain, indicates the presence of a macrophage. (H, I) raw values for CD68 + macrophage count per image frame (area of 0.15mm²) for each animal, where 15–20 (VL) or 6–8 (soleus) images were randomly selected and evaluated for a given mid-belly muscle cross-section. (H) Shows that the macrophage counts in the VL muscle increased in chow-fed males compared to chow-fed females and increased the HFS-fed males compared to HFS-fed females (p < 0.05). No change in macrophage count was observed between chow-fed females and HFS-fed females (I) Shows that the macrophage response in the soleus muscle increased in the chow-fed males compared to the chow-fed females and increased in the HFS-females compared to the HFS-fed males to the HFS diet. No change was observed in macrophage count between the chow-fed male and HFS-fed male groups. Groups without a common letter are significantly different (p < 0.05).