Fig. 3

Immunohistochemical study of cauda equina. Serial sections of the CE from rats in the EAN or siponimod group on day 12 or15 p.i were stained for the expression of Iba-1 (macrophages), CD3 (T lymphocytes), or IFN-γ using a standard immunohistochemistry protocol, with hematoxylin counterstain. The bars indicate 100 µm. A Iba-1 staining of the CE from the EAN group rat exhibits dense macrophage infiltration, primarily in the vicinity of the endoneurial vessel, spreading to the entire endoneurium. B A serial section stained for CD3 revealed numerous T cells infiltrating the endoneurium, although they were fewer than the macrophages. C Iba-1 staining in the siponimod group rat shows fewer macrophage infiltration than in the EAN group rat. D CD3 staining indicates that T cells are much fewer in the siponimod group rat than in the EAN group rat. E The number of these cells in the entire cross-sections of the CE in each sample was counted, and the corresponding cross-sectional areas were measured to determine the cell density in each group. The lower density of macrophages and T lymphocytes infiltration was observed in the siponimod group rats than in the EAN group rats (**; p < 0.01: EAN vs. siponimod groups. Statistics analysis using Mann–Whitney U-test.). F. Immunostaining of IFN-γ in CE of the EAN group rat and G the siponimod group rat on day 12 p.i.. Mononuclear cells producing IFN-γ were fewer in the siponimod group rat (black arrows in F) than in the EAN group rat. Crescent-shaped IFN-γ positive cells were found in both groups (white arrows in F and G). Those were more frequent in the siponimod group. H Comparison of the density of IFN-γ expressing mononuclear cells in CE between the EAN group and the siponimod group on day 12 p.i.. Significantly fewer IFN-γ-expressing cells were observed in the siponimod group. (*p < 0.05: EAN vs. siponimod group. Statistics analysis using Mann–Whitney U-test.)