Fig. 3

Neuronal overexpression of CARS activates microglia and the TLR2/MyD88 pathway in the hippocampus. a Representative images of Iba1 immunostaining (red) and 3D reconstruction (gray) of microglia in the hippocampus of the naïve C57BL/6J mice treated with AAV-hSyn-CARS-EGFP (CARS-overexpression or overexpression) or AAV-hSyn-EGFP (control). The areas indicated with dotted lines are magnified and shown in the “Iba1: zoom” images. b Quantification of Iba1 fluorescence intensity in the hippocampus of the control and CARS-overexpressing mice. c–f Imaris-based automated quantification of Iba1⁺ microglial filament length (c), filament area (d), filament volume (e), and numbers of dendrite branch points (f) in the hippocampus of the control and the CARS-overexpressing mice. g Sholl analyses of microglial morphology in the control and the CARS-overexpressing mice. h Representative images of immunostaining for Iba1 (blue) and TLR2 (red) in the hippocampus of the control and the CARS-overexpressing mice. i, j Representative western blotting images (i) and quantification (j) of TLR2, MyD88 and p-NF-κB protein levels in the hippocampus of the control and the CARS-overexpressing mice. k, l Representative western blotting images of p-AKT, p-JNK, p-ERK and p-P38 proteins (k) and quantification (l) of the ratio of p-AKT/AKT, p-JNK/JNK, p-ERK/ERK and p-P38/P38 in the hippocampus. m, n Representative western blotting images (m) and quantification (n) of IL-6, TNF-α, IL-1β, and IL-10 protein levels in the hippocampus. Data are shown as mean ± SEM; *P < 0.05, **P < 0.01, ****P < 0.0001