Low-Intensity Focused Ultrasound Alleviates Chronic Neuropathic Pain-Induced Allodynia by Inhibiting Neuroplasticity in the Anterior Cingulate Cortex

Abstract

Low-intensity focused ultrasound (LIFU) is a potential noninvasive method to alleviate allodynia by modulating the central nervous system. However, the underlying analgesic mechanisms remain unexplored. Here, we assessed how LIFU at the anterior cingulate cortex (ACC) affects behavior response and central plasticity resulting from chronic constrictive injury (CCI). The safety of LIFU stimulation was assessed by hematoxylin and eosin (H&E) and Fluoro-Jade C (FJC) staining. A 21-day ultrasound exposure therapy was conducted from day 91 after CCI surgery in mice. We assessed the 50% mechanical withdrawal threshold (MWT₅₀) using Von Frey filaments (VFFs). The expression levels of microtubule-associated protein 2 (MAP2), growth-associated protein 43 (GAP43), and tau were determined via western blotting (WB) and immunofluorescence (IF) staining to evaluate the central plasticity in ACC. The regions of ACC were activated effectively and safely by LIFU stimulation, which significantly increased the number of c-fos-positive cells (P < 0.05) with no bleeding, coagulative necrosis, and neuronal loss. Under chronic neuropathic pain- (CNP-) induced allodynia, MWT₅₀ decreased significantly (P < 0.05), and overexpression of MAP2, GAP43, and tau was also observed. After 3 weeks of treatment, significant increases in MWT₅₀ were found in the CCI+LIFU group compared with the CCI group (P < 0.05). WB and IF staining both demonstrated a significant reduction in the expression levels of MAP2, GAP43, and tau (P < 0.05). LIFU treatment on ACC can effectively attenuate CNP-evoked mechanical sensitivity to pain and reverse aberrant central plasticity.

Figure 1

(a) Procedure used for our experiments. Dps: days postsurgery. (b) Schematic diagram of low-intensity focused ultrasound (LIFU) stimulation.

Figure 2

Safety evaluation of LIFU stimulation (×400). Scale bars, 100 μm and 20 μm. (a) Hematoxylin and eosin (H&E) staining showed no evidence of edema, hemorrhage, or cell necrosis; n = 3 per group. (b, c) Fluoro-Jade C (FJC) staining showed that there was no significant difference in the number of FJC-positive cells between the LIFU(-) and LIFU(+) groups. Each symbol represents the mean ± SEM; independent-sample t-tests; n = 3 mice per assay.

Figure 3

Expression of c-fos (a, b) in the anterior cingulate cortex (ACC) after low-intensity focused ultrasound (LIFU) stimulation. LIFU activated neurons in the ACC (immunofluorescence (IF), ×400). Scale bar, 20 μm. ^∗∗∗P < 0.0001. Each symbol represents the mean ± SEM; unpaired t-tests; n = 3 rats per assay.

Figure 4

Effects of LIFU stimulation on the ACC in chronic neuropathic pain (CNP) model mice. 50% mechanical withdrawal threshold (MWT₅₀) significantly decreased in the CCI and CCI+LIFU groups after CCI surgery when compared with the sham group until the end of the study. After 21 days of LIFU treatment, the MWT₅₀ increased when compared with the CCI group. Each symbol represents the mean ± SEM; ^#P < 0.05 compared with the CCI and CCI+LIFU groups; ^∗P < 0.05 compared with the sham group. Two-way repeated-measures ANOVA, followed by the Bonferroni test; n = 10 per group.

Figure 5

Western blotting (WB) analysis of microtubule-associated protein 2 (MAP2) (a, b), growth-associated protein 43 (GAP43) (c, d), and tau (e, f) expression in the ACC in different groups after 21 days post-LIFU treatment. Values were normalized to β-tubulin. Each symbol represents the mean ± SEM; ^∗P < 0.05, ^∗∗P < 0.01, and^∗∗∗P < 0.001. One-way ANOVA; n = 5 rats per assay.

Figure 6

Expression of MAP2 (a), GAP43 (c), and tau (e) in the ACC of mice in different groups (immunofluorescence (IF), ×400). Scale bar, 20 μm. The expression of MAP2 (b), GAP43 (d), and tau (f) in the ACC of mice in different groups after 21 days of LIFU treatment, as detected by IF. Each symbol represents the mean ± SEM; ^∗P < 0.05,  ^∗∗P < 0.01, and^∗∗∗P < 0.001. One-way ANOVA; n = 5 rats per assay.