Fig 2: Microglia activation and increase in proinflammatory cytokine levels after AQP4 immunization.(A) Immunostaining for Iba-1 in the spinal cord of naive, pEmpty(C/P+), pAQP4(C/P–), and pAQP4(C/P+) mice. Scale bar: 50 μm. Original magnification, ×400 (inset). (B) Quantification of Iba-1 immunofluorescence intensity. (C) Coimmunostaining for Iba-1 and GFAP. (D–F) ELISA of TNF-α, IL-1β, and IL-6 levels in the spinal cord homogenate of naive, pEmpty(C/P+), pAQP4(C/P–), and pAQP4(C/P+) mice. Images are representative photomicrographs showing the ventrolateral white matter of cervical spinal cord cross sections from 5 mice per group. Nuclei were counterstained with DAPI. Data are mean ± SEM; n = 5 per group (B), n = 3 per group (D–F). ***P < 0.001, 1-way ANOVA with post hoc Tukey’s test. Scale bar: 50 μm.

Fig 3: IgG infiltration and astrocytopathy after AQP4 immunization.(A) Immunostaining for ZO-1 in the spinal cord of naive, pEmpty(C/P+), pAQP4(C/P–), and pAQP4(C/P+) mice. Insets are higher-magnification photomicrographs showing the pattern of ZO-1 staining in blood vessels. Original magnification, ×400 (insets). (B) Immunostaining for mouse IgG in the spinal cord of naive, pEmpty(C/P+), pAQP4(C/P–), and pAQP4(C/P+) mice. Dotted line demarcates the area of mouse IgG immunoreactivity. (C) Coimmunostaining for AQP4 and GFAP in the spinal cord of naive, pEmpty(C/P+), pAQP4(C/P–), and pAQP4(C/P+) mice. (D) Quantification of mouse IgG infiltration. (E and F) Quantification of AQP4 and GFAP immunofluorescence intensities. Images are representative photomicrographs showing the ventrolateral white matter of cervical spinal cord cross sections from 5 mice per group. Nuclei were counterstained with DAPI. Data are mean ± SEM; n = 5 per group. ***P < 0.001, 1-way ANOVA with post hoc Tukey’s test. Scale bars: 50 μm.

Fig 4: Oligodendrocyte loss, demyelination, and axonal loss after AQP4 immunization.(A–C) Immunostaining for Olig2 (A), MBP (B), and NF-H (C) in the spinal cord of naive, pEmpty(C/P+), pAQP4(C/P–), and pAQP4(C/P+) mice. (D) Merged images of MBP and NF-H coimmunostaining. (E and F) Quantification of Olig2 and MBP immunofluorescence intensities. (G) Quantification of the number of NF-H spots. Images are representative photomicrographs showing the ventrolateral white matter of cervical spinal cord cross sections from 5 mice per group. Nuclei were counterstained with DAPI. Data are mean ± SEM; n = 5 per group. **P < 0.01, ***P < 0.001, 1-way ANOVA with post hoc Tukey’s test. Scale bar: 50 μm.

Fig 5: AQP4 immunization induces motor impairments.(A and B) Beam walking test measuring time taken by a mouse to cross and number of paw slips while crossing a 1.2 × 80 cm (width × length) beam. (C and D) Time taken to cross and number of paw slips while crossing a 0.6 × 80 cm beam. Data are mean ± SEM; n = 8 per group. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA with post hoc Tukey’s test.