Fig. 4: LI-cTBUS suppresses reactive astrogliosis and astrocytic GABA production in vivo and in vitro. | Nature Communications

Fig. 4: LI-cTBUS suppresses reactive astrogliosis and astrocytic GABA production in vivo and in vitro.

From: Harnessing theta-gamma coupled brainwaves using ultrasound for spinal astrocyte revitalization and sustained neuropathic pain relief in mice

Fig. 4

a Experiment schematic: Lumbar-sacral spinal cords were collected after 10 consecutive days of LI-cTBUS-200 Hz. b Representative GFAP immunostaining of lumbar spinal cords at the acute phase (day 7), chronic phase (day 17), and after 10 days of LI-cTBUS. Scale bar: 200 µm. c Top: 3D reconstructions of dorsal-horn astrocytes. Scale bar: 20 µm. Bottom: Average GFAP area and dendrite volume analysis. N = 3–6 per group (control, PCI d7, PCI = 3, PCI + LI-cTBUS = 6); **p = 0.004, ***p = 0.000000018, two-way and one-way ANOVA with Tukey’s multiple comparison test. d Representative images of astrocytic GABA in ipsilateral spinal dorsal horn under LI-cTBUS stimulation. Scale bar: 20 and 200 µm. Percentage of GFAP⁺/GABA⁺ cells in PCI vs. PCI + LI-cTBUS groups. N = 6. *p = 0.03, Unpaired two-tailed t-test. e Schematic of in-vitro ultrasound stimulation of primary spinal astrocytes. f Immunocytochemistry of GFAP and GABA in cultured astrocytes. Scale bar: 10 µm. g IMARIS reconstruction of cultured spinal astrocytes. Scale bar: 20 µm. h Statistical analysis of spinal astrocyte dendrite volume (µm3). n = 10–18 per group (control = 14, control + cTBUS = 18, H2O2 200 µM = 12, H2O2 200 µM + cTBUS = 17, H2O2 400 µM = 10, H2O2 400 µM + cTBUS = 16), ***p = 0.000069, one-way ANOVA with Tukey’s multiple comparison test. i Quantification of GFAP⁺/GABA⁺ cells after H₂O₂ (400 µM) and LI-cTBUS (2.4 or 2.8 W/cm²) applied once daily for three consecutive stimulations (see the “Methods” section), n = 15. j Electron-microscopy images of endoplasmic reticulum (ER) in control, H2O2-only, and H2O2 + cTBUS-treated astrocytes. Scale bar: 5 μm. Right: ER area quantification. Data represent mean ± SEM. n = 100 recorded cells from their independent replicates. ***p = 0.0000000175, one-way ANOVA with Tukey’s multiple comparison test. k Electron-microscopy images of lysosomes. Scale bar: 2 μm. Right: Quantification of lysosome number per cell. n = 5 recorded cells from there independent replicates; **p = 0.0025, ***p = 0.000188, one-way ANOVA with Tukey’s multiple comparison test. All data are presented as box-and-whisker plots. N number of mice, n number of recorded cells. Schematic illustrations were created in BioRender. Sung, Y. (2025) https://BioRender.com/wbn112g.

Back to article page