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. 2021 Apr 13:2021:6659668.
doi: 10.1155/2021/6659668. eCollection 2021.

LIFU Alleviates Neuropathic Pain by Improving the KCC2 Expression and Inhibiting the CaMKIV-KCC2 Pathway in the L4-L5 Section of the Spinal Cord

Ye-Hui Liao  1 , Bin Wang  1 , Mo-Xian Chen  1 , Yao Liu  1 , Li-Juan Ao  1
Affiliations

LIFU Alleviates Neuropathic Pain by Improving the KCC2 Expression and Inhibiting the CaMKIV-KCC2 Pathway in the L4-L5 Section of the Spinal Cord

Ye-Hui Liao et al. Neural Plast. .

Abstract

Effective treatment remains lacking for neuropathic pain (NP), a type of intractable pain. Low-intensity focused ultrasound (LIFU), a noninvasive, cutting-edge neuromodulation technique, can effectively enhance inhibition of the central nervous system (CNS) and reduce neuronal excitability. We investigated the effect of LIFU on NP and on the expression of potassium chloride cotransporter 2 (KCC2) in the spinal cords of rats with peripheral nerve injury (PNI) in the lumbar 4-lumbar 5 (L4-L5) section. In this study, rats received PNI surgery on their right lower legs followed by LIFU stimulation of the L4-L5 section of the spinal cord for 4 weeks, starting 3 days after surgery. We used the 50% paw withdraw threshold (PWT50) to evaluate mechanical allodynia. Western blotting (WB) and immunofluorescence (IF) were used to calculate the expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), calcium/calmodulin-dependent protein kinase type IV (CaMKIV), phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB), and KCC2 in the L4-L5 portion of the spinal cord after the last behavioral tests. We found that PWT50 decreased (P < 0.05) 3 days post-PNI surgery in the LIFU- and LIFU+ groups and increased (P < 0.05) after 4 weeks of LIFU stimulation. The expression of p-CREB and CaMKIV decreased (P < 0.05) and that of KCC2 increased (P < 0.05) after 4 weeks of LIFU stimulation, but that of p-ERK1/2 (P > 0.05) was unaffected. Our study showed that LIFU could effectively alleviate NP behavior in rats with PNI by increasing the expression of KCC2 on spinal dorsal corner neurons. A possible explanation is that LIFU could inhibit the activation of the CaMKIV-KCC2 pathway.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Outline of the current view on the roles of the p-ERK–KCC2 and CaMKIV–KCC2 signaling pathways after PNI in the induction of NP. Under normal conditions, KCC2 extrudes intracellular Cl− ions from the cell and maintains the inhibitory effect mediated by GABA receptor. PNI activates TrkB and then obstructs the translation of KCC2 through the p-ERK–KCC2 and CaMKIV–KCC2 signaling pathways.
Figure 2
Figure 2
(a) Timeline of experimental protocol. Dps: days postsurgery. (b) Therapeutic effects of LIFU stimulation on NP in PNI rats. Mechanical allodynia (PWT50) was significantly decreased in the LIFU− and LIFU+ groups 3 days after PNI surgery compared with the normal and sham groups. After 3 weeks of LIFU treatment, PWT50 increased compared with the LIFU− group. Each symbol represents the mean ± SEM; ∗∗∗P < 0.001 against the LIFU− and LIFU+ groups, #P < 0.05 against the LIFU− group. One-way ANOVA; n = 10 per group.
Figure 3
Figure 3
H&E staining showed that LIFU was safe for stimulating the spinal cord ((a) ×40, scale bar = 1 mm; (b) ×100, scale bar = 200 μm) L4–L5 section of the spinal cord, showing no edema, hemorrhage, or cell necrosis.
Figure 4
Figure 4
WB analysis of KCC2 (a, b), p-ERK1/2 (c, d), CaMKIV (e, f), and p-CREB (g, h) expression in the L4–L5 section of the spinal cord in different groups at 4 weeks post-LIFU treatment. Values, normalized to β-actin, or GAPDH. Each symbol represents the mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. One-way ANOVA; n = 5 rats per assay.
Figure 5
Figure 5
Expression of KCC2 (a) p-CREB (c) in the spinal cords of rats in different groups (IF, ×400). Scale bar = 20 μm. Intensities of KCC2 (b) and p-CREB (d) IF in the spinal cords of rats in different groups after 4 weeks of LIFU treatment. Each symbol represents the mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. One-way ANOVA; n = 5 rats per assay.
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