Fig. 4.

Time-dependent activation of toll-like receptors. a On mRNA level, a significant upregulation of Tlr2 mRNA expression could be detected in the ischemic group at all analyzed time points (2 h: p = 0.004; 6 h: p = 0.007; 12 h: p = 0.004; 24 h: p = 0.015; 3 days: p = 0.002; 7 days: p = 0.003). b In contrast, Tlr3 mRNA expression level was unaltered between 2 and 24 h but was upregulated 3 (p = 0.035) and 7 days (p = 0.009) after ischemia/reperfusion. c No altered Tlr4 expression could be noted at all time points via RT-qPCR. d Exemplary images of retinal cross sections labeled with an anti-TLR3 antibody (red) and cell nuclei were counterstained with DAPI (blue). e The evaluation of the amount of TLR3⁺ cells revealed a significant higher number in ischemic retinae compared with control at all time points except 7 days (p = 0.128). Already at 2 h after ischemia, a significant difference was noted (2 h: p = 0.028; 6 h: p = 0.005; 12 h: p = 0.004; 24 h: p > 0.001; 3 days: p = 0.042). f Retinal cross sections were labeled with an anti-TLR4 antibody (red) and cell nuclei were counterstained with DAPI (blue). (G) More TLR4⁺ cells were already noted 2 h after ischemia (p = 0.020). Counts were still higher at 6 (p = 0.003), 12, and 24 h (both: p < 0.001). At 3 days (p = 0.016), an upregulation was still detectable, but not at 7 days. GCL ganglion cell layer, IPL inner plexiform layer. Values are median ± quartile + maximum/minimum for RT-qPCR and mean ± SEM for immunohistology; RT-qPCR: n = 5/group; immunohistology: n = 8/group. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar 20 μm