Fig. 5: LI-cTBUS facilitates the uptake of extracellular BDNF by spinal astrocytes.

a Left: Schematic of experimental design for flow cytometric quantification of astrocytic and microglial BDNF in the spinal cord after LI-cTBUS treatment. Middle: Representative FACS plots showing gating for astrocytes (APC-A), microglia (FITC-A), and intracellular BDNF (PE-A). Group colors: Control (Blue), PCId7 (red), PCI (yellow), and PCI+cTBUS (green). Right: Quantification shows a significant increase in BDNF-positive astrocytes in the LI-cTBUS group. Data are presented as box-and-whisker plots, *p  =  0.02, **p  =  0.007; one-way ANOVA with Tukey’s multiple comparison test, N  =  6. b Left: Experimental schematic for in vivo assessment of astrocytic uptake of exogenous BDNF-GFP protein following intrathecal injection (i.t.). Middle: Representative FACS plots show GFP-positive astrocytes (Q2: FITC-A  +  APC-A). Right: LI-cTBUS stimulation increased the percentage of double-positive cells. Data are presented as box-and-whisker plots, **p =  0.04; unpaired two-tailed t-test, N  =  4 (BDNF-GFP control), N = 6 (BDNF-GFP + LI-cTBUS). c Left: Schematic of in vitro assay using cultured primary spinal astrocytes transfected with BDNF-GFP or control proteins. Middle: FACS plots show elevated GFP signal in astrocytes following LI-cTBUS stimulation. Right: Quantification indicates a specific increase in BDNF-GFP uptake relative to non-GFP and SAR-GFP controls, indicating selective facilitation by LI-cTBUS. All data are presented as box-and-whisker plots, ***p =  0.000205; ns: not significant. n = 7, one-way ANOVA with Tukey’s multiple comparison test. N number of mice, n number of independent experiments. Schematic illustrations were created in BioRender. Sung, Y. (2025) https://BioRender.com/gfmovyn.