Anti-RGMa antibody alleviates symptoms in a rat model of NMO. (A) Schematic of the experimental procedures. A micro-syringe attached to a pulled micro-capillary needle was inserted 1 mm into the spinal cord and NMO-IgG 20 μg (human IgG from NMO patients) was infused at spinal level T10 (10th thoracic vertebrae). Arrows represent treatment days. (B) Comparative dose efficacy of NMO-IgG injection. Both 20 μg and 40 μg NMO-IgG treatment induces pathological features. n = 4. (C) Mean onset of symptoms and (D) maximum clinical score. (E) Longitudinal spinal cord section from the NMO-injected rat. Note the lack of GFAP expression only within the lesion site (*). Scale bars: 1 mm for the low-magnification images; 50 μm for the high-magnification images. (F) Specificity of anti-RGMa Ab was confirmed by western blot. (G–I) Average onset day (G) and average maximum clinical score (H) for NMO model rats given Control IgG or anti-RGMa Ab. Clinical disease score in NMO-IgG injected rats treated with either Control-IgG or anti-RGMa Ab (I). n = 10 Statistical analyses were performed using two-way ANOVA followed by Bonferroni test for B and I, and an unpaired two-tailed t test for C, D, G, and H. ns = not significant, ^* p < 0.05 ^** p < 0.01, ^*** p < 0.001. Error bars represent the SEM.

Anti-RGMa antibody ameliorates the loss of astrocyte in a rat model of NMO. (A–K) Colocalization of AQP4 and GFAP at the thoracic level of rat spinal cord tissue. (A,D,G). Representative fluorescence-microscopy images. Boxes are expanded in (B,C,E,F,H, and I). (A–C) Double labeling of AQP4 and GFAP was observed along the entire glia limitans, astrocytic processes, and end-feet (arrows in B), around capillaries and encircling of the blood vessels (arrows in C) in non-NMO control rats. (D–F) Co-expression of AQP4 and GFAP was lost in NMO rats that did not receive anti-RGMa mAb. Arrowheads represent the loss of co-staining along glia limitans I. Colocalization around capillaries and encircling of the blood vessels in non-NMO rats (arrows in C) was virtually absent in control-IgG-NMO rats (F). The arrowheads in F indicate expression of the few remaining astrocytes. (G–I) AQP4 and GFAP double-positive cells were increased in NMO rats treated with anti-RGMa mAb as compared to Control-IgG-treated NMO rats. Arrows indicate the double staining of AQP4 and GFAP in the dorsal white matter (H), astrocyte processes, and endfeet (I). (J,K) Quantitative analysis of fluorescence intensity of AQP4-(J) and GFAP- (K) positive cells from the high magnification images (B,C,E,F,H,I). Statistical analysis was performed by ANOVA followed by Bonferroni test for J and K. ns = not significant, ^* p < 0.05, ^** p < 0.01. Error bars represent SEM. Scale bars: 200 μm (low magnification images in A,D,G), 100 μm (high magnification images in (B,C,E,F,H,I). (L) Representative images of MBP expression, a marker for myelin. MBP expression did not display any obvious differences among the groups. Scale bar: 500 μm.

Cellular infiltration of Iba1⁺ and CD45⁺ cells in NMO rats. (A–H) Microscopic images of fluorescence intensity in rat spinal cord sections at the site of injury in order to show the distribution of Iba1⁺ cells (A–C) or CD45⁺ cells (E–G). Quantitative analysis of fluorescence intensity showed that anti-RGMa reduced the infiltration of Iba1⁺ cells (D) and CD45⁺ cells (H) at the injury site. Statistical analysis was performed by ANOVA followed by the Bonferroni test. ^* p < 0.05, ^** p < 0.01. Error bars represent SEM. Scale bars: 100 μm (A–C), 50 μm (D–F).

Anti-RGMa antibody reduces IL17A⁺ T-cell infiltration in a rat model of NMO. (A–C) Flow cytometry sorting of CD11b⁺ CD45^low (microglia) and CD11b⁺ CD45^hi (macrophage) from rat spinal cords at injury sites. In all groups, microglia were detected, but very few or no macrophages were detected. (D–H) Flow cytometry gating strategy of T-cells (CD3⁺, CD45⁺; D–F). Higher IL17A⁺ T-cell density was observed in Control-IgG-treated NMO rats (G) than in NMO rats injected with anti-RGMa mAb (H).

Anti-RGMa antibody reduces axonal loss in a rat model of NMO. (A–F) Representative confocal images of SMI-312-positive axonal staining in the dorsal column of rat spinal cords. Boxes are expanded in (B,D, and F). Number of axons was significantly less in Control-IgG-NMO rats (C,D) when compared to non-NMO rats (A,B). However, SMI-312 labeled axons were preserved in the NMO rats that received anti-RGMa mAb (E,F). (G) Quantitative analysis of preserved axonal counts (SMI-312⁺ axons/mm²) from A, C, and E (n = 3). Statistical analysis was performed using ANOVA followed by the Bonferroni test. ^* p < 0.05, ^** p < 0.01. Error bars represent SEM. Scale bars: 150 μm for low-magnification images (A,C,E,); 20 μm for high-magnification images (B).