Fig. 3

NMO–IgG intracerebral injection induced AQP4 loss and enhanced expression of BTK and pBTK in microglia. A Experimental NMO mice were induced with NMO–IgG and complement. The mice were administered zanubrutinib (5, 10, 20 mg/kg) for 5 days. B Immunofluorescent staining of BTK protein expression in the brains of mice either treated with control–IgG or NMO–IgG. C Numbers of BTK⁺ Iba-1⁺ microglia were analyzed in control–IgG recipient mice (control–IgG) and NMO–IgG mice. D MRI presentation in different groups. T2-weighted images showed lesion areas dotted with yellow dashed lines, The blue lines represent the needle tract. E Quantification of the lesion volume induced by NMO–IgG after receiving BTKi treatment or not. F Immunofluorescent staining of AQP4 at 5 days after injection. The yellow lines delimit the lesion with AQP4 loss. Scale bar is 200 μm. G Quantification of the lesion size induced by NMO–IgG after receiving BTKi treatment or not. H Colocalization of astrocytic and endothelial cells in the absence or presence of zanubrutinib. Astrocytes were stained with AQP4 (red), and endothelial cells were stained with CD31 (green). I Quantification of relative length of CD31⁺ parenchymal blood vessels covered by AQP4 around NMO–IgG induced lesion (n = 6 per group). J Immunofluorescence staining of phosphorylated BTK (Y223) (pBTK) expressed on microglia in the absence or presence of zanubrutinib. K Numbers of pBTK⁺ Iba-1⁺ microglia were analyzed 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 per group). The NMO–IgG + BTKi represented NMO–IgG recipient mice with the treatment of 20 mg/kg zanubrutinib, for no significant statistical differences were found when the zanubrutinib dosages were 5 or 10 mg/kg (data not shown). Scale bars, 100 μm. Data are displayed as means ± SEM. Statistical analysis was conducted by one-way ANOVA or Student’s t test