Figure 4.

Treatment with vitamin B complex induces time-dependent changes of Ca_V1.2 channel expression in M1 macrophages after PNI. To evaluate cellular distribution of the Ca_V1.2 isoform of l-VDCCs (green), cross sections of femoral nerve obtained from the: (A) operated (O); and (B) operated and treated with vitamin B complex (OT) groups were counterstained with anti-TNF-α (M1 marker, blue) and anti-ED1 (red) antibodies. The quantification of single-, double-, and triple-positive cells is presented as number of ED1⁺ cells/mm², ED1⁺/TNF-α⁺ cells/mm², and ED1⁺/TNF-α⁺/Ca_V1.2⁺ cells/mm² (C), and as the percentage of triple -positive cells (ED1⁺/TNF-α⁺/Ca_V1.2⁺ cells) in ED1⁺ and ED1⁺/TNF-α⁺ cell populations (D). The data are shown as the mean ± SEM of three independent experiments (three images/group/independent experiment were captured). Statistical analysis was performed using a two-sided Student’s t-test (* p < 0.05 OT vs. O group, as indicated at the graphs). Intensive Ca_V1.2 staining, besides in M1 macrophages, was observed in axons (green asterisks) and in some ED1⁻ cells (yellow and green arrows) as well. ED1⁺/Ca_V1.2⁺/TNF-α⁺ M1 macrophages are marked with a white arrowhead, ED1⁺ macrophages with oval/round morphology (M1 type) are marked with a red arrowhead, and “foamy” ED1⁺ macrophages (M2) are indicated with white arrows. # indicates where the high magnification micrographs are taken from. Scale bars: 20 µm and 100 µm. PNI: peripheral nerve injury; TNF: tumor necrosis factor.