Fig. 6: Involvement of the mechanical ion channel TRPA1 in LI-cTBUS-induced analgesia. | Nature Communications

Fig. 6: Involvement of the mechanical ion channel TRPA1 in LI-cTBUS-induced analgesia.

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

Fig. 6

a Top: Experimental paradigm showing intraperitoneal (i.p.) injection of the TRPA1 inhibitor HC030031. PCI mice received HC030031 on day 17 post-injury, followed by LI-cTBUS stimulation one hour later. The procedure was repeated for 8 days. Bottom: The 50% withdrawal threshold revealed that HC030031 partially inhibited LI-cTBUS-induced analgesia in PCI mice. Data are presented as mean ± SEM, *p = 0.04, **p = 0.002, ***p = 0.00018, two-way ANOVA with Tukey’s multiple comparison test, N = 9. b Top: Schematic of fiber photometry recording in the spinal dorsal horn of Thy1-Gcamp6f mice. Scale bar: 200 µm. Bottom: Calcium responses in the spinal dorsal horn were reduced in HC030031-injected mice. Area under the curve analysis confirmed a significant reduction; data are presented as box-and-whisker plots, ***p = 0.0000000853, two-tailed t-test, repeated measurements. n = 26. c Top: Paw withdrawal threshold (PWT) in PCI wild-type and TRPA1 KO PCI mice treated with LI-cTBUS from day 17 to day 26 post-injury, data are represented as mean ± SEM. Bottom: Area under the curve of PWT over the stimulation period. Data are presented as box-and-whisker plots, **p = 0.006, ***p = 0.00044, one-way ANOVA with Tukey’s multiple comparison test, N = 6. d Left: Representative GFAP immunostaining in the spinal dorsal horn of PCI wild-type and TRPA1 KO PCI with and without LI-cTBUS treatment. Spinal cord samples were collected on day 26 following 10 days of stimulation. Scale bar: 200 µm. (Middle) 3D reconstruction of astrocyte morphology in each group. Scale bar: 20 µm. Right: Quantification of astrocyte filament number (number of astrocyte processes) and average dendrite volume. Data are presented as box-and-whisker plots, *p = 0.02, **p = 0.005, ***p = 0.00000001, one-way and two-way ANOVA with Tukey’s multiple comparison test. ns: non-significant, p > 0.99. N = 3-6 per group (WT PCI, WT PCI + LI-cTBUS = 6, TRPA1-KO PCI = 3, TRPA1-KO PCI + LI-cTBUS = 4). e Top: Immunostaining of GFAP and GABA in spinal dorsal horn astrocytes of wild-type PCI and TRPA1 KO PCI with and without cTBUS stimulation. Scale bar: 20 µm. Bottom: Quantification of GFAP/GABA positive cells, n = 4–6 per group (WT PCI, WT PCI + LI-cTBUS = 4, TRPA1-KO PCI, TRPA1-KO PCI + LI-cTBUS = 6). Data are presented as box-and-whisker plots, *p = 0.03, **p = 0.001; ns: not significant. One-way ANOVA with Tukey’s multiple comparison test. f Schematic of TRPA1 agonist (NMM) treatment in H₂O₂-exposed primary spinal astrocyte cultures. Representative immunostaining of GFAP and GABA. Right: Quantification of GFAP+/GABA+ cell proportion and GABA intensity, N = 9. Data are presented as box-and-whisker plots, *p = 0.05, **p = 0.007, ***p = 0.0003; ns: not significant. 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/bf9u5xm.

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