Fig. 3

(A-L) 20x-magnification cross-sectional images of lesioned spinal cords in the Shh- and Control group 3- and 7-days post injury (dpi) stained for CD206 (red; A-D), iNOS (green; E-H), and composite images (I-L; scale bar: 150 μm). (M-P) The corresponding 10x-magnification 4 × 4 tile-scans are presented as composite images, with the overlying inlets (A-L) depicted in red (scale bar: 500 μm). Due to the fluid nature of M(IFN-γ) to M(IL-4) polarization, distinguishing between the expression of CD206 and iNOS remains challenging. Therefore, iNOS-expressing cells without CD206 co-staining (iNOS⁺/CD206⁻) were counted as indicative of M(IFN-γ)-polarized macrophages, while iNOS⁻/CD206⁺ cells were considered indicative of M(IL-4)-polarized macrophages. (Q) While no significant difference was observed at 3 dpi, Shh treatment (n = 5) was associated with significantly fewer M(IFN-γ)-polarized, pro-inflammatory macrophages compared to untreated controls at 7 dpi (p < 0.001, n = 6). (R) Simultaneously, an increase in M(IL-4)-polarization towards an anti-inflammatory macrophage phenotype was noted under Shh treatment (n = 5) compared to untreated animals (p < 0.001, n = 6). Data are expressed as mean ± SEM cells/mm². Statistical analysis was performed using one-way ANOVA with post hoc Tukey-HSD tests (***p < 0.001)