The WN1316 treatment suppresses astrocytic and microglial activation and reduces the level of IL-1β and iNOS in the spinal cord.

<p>(A) Immunohistochemical analysis of paraffin embedded sections of the lumbar spinal cord from non-Tg (a, d, and g), vehicle-treated ALS(SOD1^H46R) (b, e, and h), and 10 µg/kg WN1316-treated ALS(SOD1^H46R) (c, f, and i) mice at 21–22 weeks of age (late symptomatic stage). Representative images of immunostaining with GFAP (a, b, and c) and Iba-1 (d, e, and f) and histochemical staining with hematoxylin-eosin (H&E) (g, h, and i) are shown. Scale bar indicates 100 µm. (B) Immunohistochemical analysis of frozen sections of the lumbar spinal cord from non-Tg, vehicle-treated ALS(SOD1^H46R), and 10 µg/kg WN1316-treated ALS(SOD1^H46R) mice at 21–22 weeks of age (late symptomatic stage). Representative merged images of double immunostaining with IL-1β (green) and GFAP (for astrocytes) (red), Iba-1 (for microglia) (red), or microtubule-associated protein 2 (MAP2) (for neurons) (red) are shown. Merged signals are shown in yellow. Scale bar indicates 50 µm. (C) Western blot analysis of the iNOS protein in the lumbar spinal cord from ALS(SOD1^H46R) mice treated with vehicle or 10 µg/kg WN1316 at 21–22 weeks of age (late symptomatic stage) and from age-matched non-Tg littermates, and quantitative analysis of iNOS-immunoreactive bands. The NP-40-soluble fraction (2 µg) was used for immunoblotting with anti-iNOS antibody. Glyceraldhyde-3-phosphate dehydrogenase (GAPDH) was used as control for NP-40-soluble fraction. Densitometric data for iNOS-immunoreactive bands was normalized by the level of GAPDH. Data are expressed as mean ± SEM (n = 4). * <i>p</i><0.05, ** <i>p</i><0.01 by one-way ANOVA with Tukey’s <i>post hoc</i> test.