Figure 7. Impact of GTE- and cocoa-supplemented diets on the aging-associated microgliosis and imbalance in M1/M2 microglial phenotypes found in the ventral horn spinal cord of old mice. Lumbar spinal cord sections were immunostained for the microglial marker Iba1 (red), and either Mac-2 or CD206 (green), for M1 or M2 microglia, respectively; fluorescent Nissl staining (blue) was used for MN visualization. (A) Quantification of microglia expressed as the percentage of ventral horn occupied by Iba1-positive profiles. (B1–D2) Representative confocal images showing Iba1-staining around spinal cord MNs of animals from control, GTE and cocoa groups as indicated in panels. (E, F, J, K) Quantification of Mac-2-positive (E) and CD206-positive (J) profiles surrounding MNs shown as the percentage of ventral horn area occupied by the immunostained profiles; the proportion of microglial profiles expressing both Iba1 and either Mac-2 (F) or CD206 (K) is also shown. The average values of these parameters in adult mice from our previous study  are indicated in each graph (red lines) for comparison purposes. (G1–I4, L1–N4) Representative confocal micrographs used for data analysis showing Mac-2 (G1–I4) and CD206 (L1–N4) in combination with Iba1 and fluorescent Nissl staining, as indicated in panels. Data in the graphs are expressed as the mean ± SEM; a total of 45-50 images per experimental group were analyzed (number of animals per group: control [Ctrl] = 3, GTE = 4, cocoa = 5); *p < 0.05 and ***p < 0.001 vs. Ctrl (one-way ANOVA, Bonferroni's post hoc test). Scale bar in N4 = 50 μm (valid for B1–D1, G1–I4, L1–N3).