Figure 3. NSCs and 3K3A-APC combination therapy enables anatomical and functional improvements of dMCAo disrupted neural circuitry.

(a) Viral-labeled axons projecting from the primary motor cortex (M1) to the somatosensory cortex (S1) 35 d after dMCAo. WM, white matter. Arrows, direction of axonal projections. Dotted lines, ischemia-affected border in the S1 region. Scale bar = 50 μm. (b) Tuj1⁺ NSCs and viral-labeled axonal projections (AAV.TurboRFP) in the S1 boxed regions magnified from a. Arrowheads, TurboRFP⁺ axons and Tuj1⁺ NSCs points of contact. Scale bar = 50 μm. (c) 3D reconstruction of a point of contact in the boxed region (7 μm x 7 μm x 7 μm) magnified from b. Scale bar = 50 μm. (d) Scatter plot of TurboRFP⁺ axons and Tuj1⁺ NSCs contacts from b–c. n = 5 mice/group; significance by Student’s-t test. (e) Synaptophysin (red; presynaptic marker) and PSD-95 (green; postsynaptic marker) co-localized puncta on SC121⁺ NSCs in the S1 region (magenta). Arrows, co-localized puncta. Insets, high magnification of boxed regions showing co-localized puncta. DAPI (blue), nuclear staining; Scale bar = 5 main images, 1 μm insets. Dashed lines, borders between NSCs. (f) Scatter plot of synaptophysin and PSD-95 co-localized puncta on SC121⁺ cells in the S1 region 35 d after dMCAo. n = 5 mice/group; significance by Student’s-t test. (g) Voltage-sensitive dye (VSD) imaging of cortical responses to forelimb stimulation in NSCs and vehicle-treated (top) or NSCs and 3K3A-APC-treated (bottom) mice 35 d after dMCAo. Scale bar = 0.5 mm. FL, the forelimb somatosensory cortex. (h–j) VSD signal responses in the FL cortex (h), scatter plot of peak VSD amplitude (i) and time to peak response (j). n = 5 mice per group, significance by one-way ANOVA and Bonferroni’s post hoc test.