Fig 2: 3-NT immunofluorescence in the canine frontal cortex. The frontal cortex of a 16-year-old dachshund with canine cognitive dysfunction is shown. 3-NT highly immunoreactive cells were detected in and around Aβ plaques (A–C), and parenchymal 3-NT staining was observed (B,C). Cerebral amyloid angiopathy (CAA)-affected blood vessel (D,F) showed 3-NT staining (E,F,H,I). 3-NT was highly expressed in GFAP-positive astrocytes (G–I,J–L), clustered around blood vessel walls (G–I). 3-NT was detected in neuronal cytoplasm and nuclei (N,O) [NEUN marks neurons (M,O)] and neuronal process (Q,R) [NFH marks neurons (P,R)]. Images (C F,I,L,O,R), are merged images of two preceding images in the same line. Nuclei were counterstained with DAPI. Scale bars are 5 μm in (D–F) and (J–L), 20 μm in (P–R), and 10 μm in (A–C,G–I,M–O).

Fig 3: Pazopanib reduces spinal NF-kB-glial axis activation. Spinal cords (L3-L5) were assessed for the expression of pNF-κB and VEGFA and the reactivity of astrocytes and microglia using double immunofluorescence (IF). IF assays were performed in histological sections of spinal cord tissues in mice at 12 weeks post-partial medial meniscectomy (PMM). IA injection of pazopanib (Paz), vandetanib (Van) or vehicle (Veh, 5% DMSO in PBS) was performed at week 1 (Gp1, inflammatory pain stage), week 4 (Gp2, early OA stage) or week 8 (Gp3, advanced OA stage) after PMM, twice per week for 12 weeks, and the effect of pazopanib, vandetanib or vehicle on the expression of pNF-κB and VEGFA and the reactivity of astrocytes and microglia in the lumbar spinal dorsal horns were observed; the expression of GFAP (an astrocyte marker, red), IBA1 (a microglia marker, green), pNF-κB (red) and VEGFA (green) was examined by IF microscopy (A). Quantitative analysis demonstrated that the expression of GFAP, IBA1, VEGFA and NF-κB activation were significantly increased after PMM (n=3) (B-M). Statistical analysis was conducted using one-way ANOVA followed by the Tukey-Kramer test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 comparisons between groups with or without pazopanib or vandetanib treatment in mice that underwent PMM. 4′,6-diamidino-2-phenylindole (DAPI) stains nuclei blue. Scale bars: 100 μm.

Fig 4: Experimental design. (a) Schematic representation of the experimental design. (b) E16.5 mouse cortical neurons were cultured in vitro for 7 days and double stained with the neuronal marker Tuj1 (green) and glial marker GFAP (red). Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.

Fig 5: nNOS immunofluorescence in the canine frontal cortex. The frontal cortex of a 16-year-old dachshund with canine cognitive dysfunction is shown. nNOS cytoplasmic staining was detected in neuronal somas and processes around Aβ plaques (A–C). Neurons labeled by TUBB3 (D) and NEUN (G) were immunoreactive for nNOS in the superficial cortical layers (D–F) and deep cortical layers (G–I). Some astrocytes, marked by GFAP (J), were immunopositive for nNOS (K,L). The third column shows merged images of the first two images of each line. Nuclei were counterstained with DAPI. Scale bars are 10 μm in (A–C), 20 μm in (D–F), and 5 μm in all other images.