Fig. 5

BTK inhibition attenuated microglia and astrocyte recativity as well as their interaction in NMO mice. Immunofluorescent staining of microglia (A) and astrocytes (B) in the absence or presence of zanubrutinib (20 mg/kg). Activated microglia were stained with CD86 (green) and Iba1 (red). Reactive astrocytes were immune-stained with GFAP (green) and complement C3 (red). Quantification of the numbers of Iba1⁺ microglia (C), Iba1⁺ CD86⁺ microglia (D), GFAP⁺ astrocytes (E) and GFAP⁺ C3⁺ astrocytes (F) in the absence or presence of zanubrutinib treatment at stepwise doses (5, 10 or 20 mg/kg). n = 6 mice per group. Scale bars, 100 μm. G–I Morphological analysis of microglia in control–IgG recipient mice (control–IgG) and NMO–IgG mice in the absence (NMO–IgG) or presence (NMO–IgG + BTKi) of zanubrutinib (20 mg/kg), n = 6 mice per group. G The morphology and skeleton of microglia, Scale bars, 20 μm. H Quantification analyses of process length, endpoints number, and branch number in microglia. I Sholl analysis of microglia on day 5 after NMO–IgG mice model was induced. Data are displayed as means ± SEM. Statistical analysis was conducted by one-way ANOVA. J Interaction between Iba1⁺ microglia (green) and GFAP⁺ astrocytes (red) are inferred from the enlargement and overlapping of cells and their processes in dual immunostaining. Zanubrutinib was administered at a dose of 20 mg/kg. K Quantification of Iba1⁺ microglia and GFAP⁺ astrocytes interaction in control–IgG recipient mice (control–IgG) and NMO–IgG mice in the absence (NMO–IgG) or presence (NMO–IgG + BTKi) of zanubrutinib (20 mg/kg). n = 6 mice per group (3 sections/mouse)