Fig 1: Histological validation of astrocyte-ependymal cell subtypes and cells with only astrocyte markers, and the demonstration of cell activation induced by laminectomy in mouse spinal cord(A) Astrocyte-ependymal-Nes cells (arrow) co-expressed GFAP+ (red), SOX9+ (blue), FOXJ1+ (green), and Nestin+ (violet) in lateral column from 7 dpi (at 1,200 µm rostral from epicenter) samples.(B) Astrocyte-ependymal-Neslow cells (arrows) co-expressed SOX9 (blue) and FOXJ1 (green) in the ventral column from naive, sham, 7 dpi (at 1,200 µm rostral from epicenter), and SCI1M (at epicenter) samples.(C–E) Histological analysis illustrating the number of astrocyte-ependymal cells (C), the proportion of astrocyte-ependymal and astrocyte-ependymal-Neshigh subpopulations among tdT+ cells (D), and the number of astrocyte-ependymal-Neshigh cells (E) in naive, sham, and 7 dpi groups at 800–1,200 µm rostral and caudal from the epicenter.(F) Astrocyte-ependymal-Neshigh cells (arrows) co-expressed SOX9 (blue), FOXJ1 (green), and NES (white) in the lateral column.(G) Confocal images and 3D reconstruction of the cells with only astrocyte markers (arrows) by staining with DAPI (blue), AGT (green), and GFAP (white) in ventral column from all groups (7 dpi, at epicenter; SCI1M, at 800 µm rostral from epicenter).(H) (H1) GFAP (green) and tdT (red) expression in ventral column from naive and sham groups (at 600 µm rostral from the epicenter). (H2) Histological analysis demonstrated the number of tdT+ cells in naive, sham, and 7 dpi groups at 800–1,200 µm rostral and caudal from the epicenter. N = 3–4 mice per group. Barplots represent mean ± SEM. Independent t tests. *p < 0.05; **p < 0.01, ***p < 0.001 compared with other group; GM, gray matter; WM, white matter; Ast-Epe, astrocyte-ependymal cell; Ast-Epe-Neshigh, astrocyte-ependymal-Neshigh cell. Scale bar, 20 µm.