Figure 1.

Gsx1 expression promotes cell proliferation in the injured spinal cord

(A) Lateral hemisection SCI was performed on 8- to 12-week-old mice at the T9–T10 level immediately followed by the injection of lentivirus encoding Gsx1 along with RFP reporter (lenti-Gsx1-RFP). Lentivirus encoding only the reporter RFP was used as a control (lenti-Ctrl-RFP). Spinal cord tissues were analyzed by immunohistochemistry, RNA-seq, Ingenuity Pathway Analysis (IPA), and quantitative real-time PCR analysis. Scale bar, 100 μm. (B) Confocal images of sagittal sections of spinal cord tissue at 3 DPI show the expression of the viral reporter RFP and cell proliferation marker Ki67 (n = 3 for sham and n = 6 for SCI+Ctrl and SCI+Gsx1). Arrows indicate Ki67⁺/RFP⁺ co-labeled cells. Images in the bottom left corner show a higher magnification z stack view of the area denoted by a dashed white box. Scale bars, 20 μm. (C and D) Quantification of all Ki67⁺ cells and Ki67⁺/RFP⁺ cells. (E) Quantitative real-time PCR analysis of Ki67 mRNA expression at 3 DPI, normalized to the sham group; n = 4. Mean ± SEM. ∗p < 0.05 by Students’ t test (D) and one-way ANOVA followed by a Tukey post hoc test (C and E). (F) Box plot of genes known to promote cell proliferation (n = 3). Each dot represents the gene expression as log₂(count per million) for one biological replicate sample. (G) List of differentially expressed genes that are known to inhibit cell proliferation between SCI+Gsx1 and SCI+Ctrl groups by RNA-seq analysis (n = 3).