Figure 5.

Dynamic microglia polarization is independent of substrate elasticity. (A) Microglia, seeded on substrates of varying stiffness, were exposed to LPS for 24 h, while either receiving an anti-inflammatory preconditioning stimulus (IL-4) for 24 h (beige) or no preconditioning (blue). (B) While IL-4 preconditioning exerted a robust inhibitory effect, there was no significant effect of substrate elasticity, as assessed by iNOS mRNA expression (n = 5). (C) Representative immunocytochemical images show Iba-1 positive microglia (red) counterstained for inducible nitric oxide synthase (iNOS; green), with nuclei stained blue on substrates of 0.6 kPa without preconditioning, and (D) IL-4 preconditioned cells (scale bar = 100 mm). (E) Expression of iNOS on protein level assessed immunocytochemically (n = 3) yielded the same results as on the mRNA level. (F) Parallel to iNOS, the concentration of secreted pro-inflammatory cytokine interleukin-1b (IL-1b) within the cell medium was not affected by substrate stiffness but was downregulated significantly by the anti-inflammatory stimulus, ELISA (n = 6). (G) Likewise, IL-6 RNA expression was significantly downregulated under an anti-inflammatory preconditioning stimulus without elasticity specific changes (*p < 0.05; **p < 0.01, ***p < 0.001; n = 6).