Fig 2: MiR-127 increased neuronal loss, promoted cell apoptosis and inhibited axonal regeneration after SCT.Slices of rostral spinal cord derived from 28 dpo were subjected to immunohistochemistry for NeuN (a–d, red, white arrow), CGRP (e–h, red, white arrow), GAP-43 (i–l, red, white arrow), and counterstained with DAPI (blue) in sham group (a,e,i, n = 5), SCT group (b,f,j, n = 5), NS-miRNA (c,g,k, n = 5) and miR-127 group (d,h,l, n = 5). Three days post operation, TUNEL staining was used to analyze neuronal apoptosis (m–p, red, white arrow) in rostral of spinal cord in sham, SCT, NS-miRNA and miR-127 group. Sections were stained with DAPI (blue) to show all nuclei, and TUNEL (red, white arrow) to show apoptotic cells, in merged photomicrographs rose-red were defined as TUNEL positive. (q) The percentage of the NeuN+/DAPI was measured. (r,s) Mean density of CGRP (r) and GAP-43 (s), which presented as IOD/Area in each group were measured. (t) Quantitative histogram showed the percentage of TUNEL/DAPI in sham, SCT, NS-miRNA and miR-127 group. *P < 0.05, **P < 0.01. Scale bar: (a–d), 100 µm; (e–p), 50 µm.

Fig 3: Immunofluorescent staining for (A) nuclei (blue), (B) CK (green) and (C) CK19 (red), (D) merge picture for CK and CK19 and (E) BF control in sphere-forming cells. Immunofluorescent staining for (F) nuclei (blue), (G) NSE (green) and (H) GFAP (red), (I) merge picture for GFAP and (J) BF control in the sphere-forming cells. Immunofluorescent staining for (K) nuclei (blue), (L) NF-M (green) and (M) S100 (red), (N) merge picture for S100 and (O) BF control in the sphere-forming cells. Immunofluorescent staining for (P) nuclei (blue), (Q) GAP-43 (green) and (R) P75NTR (red), (S) merge picture for GAP-43 and P75NTR and (T) BF control in the sphere-forming cells. Magnification, ×400. DAPI, 4′,6-diamidino-2-phenylindole; CK, cytokeratin; BF, brightfield; NSE, neuron-specific enolase; GFAP, glial fibrillary acidic protein; NF-M, neurofilament medium; GAP-43, growth associated protein-43; P75NTR, p75 neurotrophin receptor.

Fig 4: Western blot and immunohistochemistry of GAP-43 in the hippocampal CA1 region and brainstem (magnification, ×200). Analysis of GAP-43 in the (A) hippocampal CA1 region and (B) brain stem at each time point following DAI (1, 3 and 7 days). Photographs of GAP-43 staining in the hippocampal CA1 region in (C) Sham, (D) DAI+Vehicle and (E) DAI+FK506 groups. Photographs of GAP-43 immunohistochemistry staining in the brainstem in (F) Sham, (G) DAI+Vehicle and (H) DAI+FK506 groups. Quantitative analysis of the GAP43 in the (I) hippocampal CA1 region and (J) brainstem in Sham, DAI+Vehicle and DAI+FK506 groups at 1, 3 and 7 days post-injury. *P<0.05 vs. Sham group; #P<0.05 vs. DAI+Vehicle group. GAP-43, growth-associated protein-43; DAI, diffuse axonal injury.

Fig 5: Colocalization of ALK5 with Nestin or DCX after I/R; ALK5 promotes axonal regeneration after I/R.a Representative images of immunofluorescence staining for ALK5 (green), Nestin (red)/ DCX (red) and cellular nuclei (blue). (scale bar = 100 µm). Arrows show the positive cells, and the inserted images show magnified images of representative cells. b Representative Western blot images of GAP-43 expression with LV-ALK5-RNAi or LV-con-RNAi injection 24 h and 14 d after I/R (n = 6 biological replicates). c Comparison of mean intensity ratios in Western blot analysis for GAP-43 with ALK5 knockdown. d Representative Western blot images of GAP-43 expression with LV-ALK5 or LV-con-ALK5 injection 24 h and 14 d after I/R (n = 6 biological replicates). e Comparison of mean intensity ratios in Western blot analysis for GAP-43 with ALK5 overexpression. *P < 0.05, compared to the LV-con-RNAi or LV-con-ALK5 group at the same time point (Student’s t test). f Comparison of the mean density value in immunohistochemical analysis for NF-200 expression. *P < 0.05, compared to the LV-con-RNAi group at the same time point; ?P < 0.05, compared to the LV-con-ALK5 group at the same time point; #P < 0.05, compared to the sham group at the same time point (ANOVA). g Representative images of immunohistochemical staining for NF-200 expression in each group 24 h and 14 d after I/R (n = 5 biological replicates). (scale bar = 100 µm)