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 ( ) 3 days post-PNI surgery in the LIFU⁻ and LIFU⁺ groups and increased ( ) after 4 weeks of LIFU stimulation. The expression of p-CREB and CaMKIV decreased ( ) and that of KCC₂ increased ( ) after 4 weeks of LIFU stimulation, but that of p-ERK1/2 ( ) 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.

Although neurocircuits can be activated by focused ultrasound stimulation, it is unclear whether this is also true for spinal cord neurocircuits. In this study, we used low-intensity focused ultrasound (LIFU) to stimulate lumbar 4–lumbar 5 (L4–L5) segments of the spinal cord of normal Sprague Dawley rats with a clapper. The activation of the spinal cord neurocircuits enhanced soleus muscle contraction as measured by electromyography (EMG). Neuronal activation and injury were assessed by EMG, western blotting (WB), immunofluorescence, hematoxylin and eosin (H&E) staining, Nissl staining, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), somatosensory evoked potentials (SEPs), motor evoked potentials (MEPs), and the Basso–Beattie–Bresnahan locomotor rating scale. When the LIFU intensity was more than 0.5 MPa, LIFU stimulation induced soleus muscle contraction and increased the EMG amplitudes ( ) and the number of c-fos- and GAD65-positive cells ( ). When the LIFU intensity was 3.0 MPa, the LIFU stimulation led to spinal cord damage and decreased SEP amplitudes for electrophysiological assessment ( ); this resulted in coagulation necrosis, structural destruction, neuronal loss in the dorsal horn by H&E and Nissl staining, and increased expression of GFAP, IL-1β, TNF-α, and caspase-3 by IHC, ELISA, and WB ( ). These results show that LIFU can activate spinal cord neurocircuits and that LIFU stimulation with an irradiation intensity ≤1.5 MPa is a safe neurostimulation method for the spinal cord.

Low-intensity focused ultrasound (LIFU) has been shown to provide effective activation of the spinal cord neurocircuits. The aim of this study was to investigate the effects of LIFU in order to alleviate spasticity following spinal cord injury (SCI) by activating the spinal neurocircuits and increasing the expression of the neuronal K-Cl cotransporter KCC₂. Adult male Sprague Dawley (SD) rats (220–300 g) were randomly divided into a sham control group, a LIFU⁻ group, and a LIFU⁺ group. The mechanical threshold hold (g) was used to evaluate the behavioral characteristics of spasm. Electromyography (EMG) was used to assess activation of the spinal cord neurocircuits and muscle spontaneous contraction. Spasticity was assessed by frequency-dependent depression (FDD). The expression of KCC₂ of the lumbar spinal cord was determined via western blot (WB) and immunofluorescence (IF) staining. The spinal cord neurocircuits were activated by LIFU simulation, which significantly reduced the mechanical threshold (g), FDD, and EMG recordings (s) after 4 weeks of treatment. WB and IF staining both demonstrated that the expression of KCC₂ was reduced in the LIFU⁻ group (P < 0.05). After 4 weeks of LIFU stimulation, expression of KCC₂ had significantly increased (P < 0.05) in the LIFU⁺ group compared with the LIFU⁻ group. Thus, we hypothesized that LIFU treatment can alleviate spasticity effectively and upregulate the expression of KCC₂ in the L4–L5 section of SCI rats.

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 ( ) with no bleeding, coagulative necrosis, and neuronal loss. Under chronic neuropathic pain- (CNP-) induced allodynia, MWT₅₀ decreased significantly ( ), 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 ( ). WB and IF staining both demonstrated a significant reduction in the expression levels of MAP2, GAP43, and tau ( ). LIFU treatment on ACC can effectively attenuate CNP-evoked mechanical sensitivity to pain and reverse aberrant central plasticity.

AIM: To explore the efficacy and possible mechanisms of Low-intensity focused ultrasound (LIFU) in alleviating spasticity caused by Spinal cord injury (SCI). MATERIAL and METHODS: We selected male Sprague-Dawley rats as subjects and performed transverse injuries on the T9 vertebra of their spinal cord (SC) to build SCI. On the 7th day after SCI, LIFU treatment was performed below the SCI segment once a day for 20 min, for 4 consecutive weeks. During treatment, a pressure sensor was used to assess the degree of spasticity. After treatment, the SC tissues from the treatment sites of the SCI+LIFU(-) and SCI+LIFU(+) groups were extracted, and high-throughput sequencing was performed to identify the changes in proteomics. In addition, expression of the growth associated protein 43 (Gap43) was validated by western blotting. RESULTS: The behavioral results suggested that after 2 weeks of SCI, the rats were significantly induced to have a spastic reaction (p<0.05), while after 4 weeks of LIFU treatment, the spastic response of rats was significantly improved (p<0.05). Western blot analysis showed a significant increase in Gap43 expression in the SCI+LIFU(-) group compared with the sham group, whereas after 4 weeks of LIFU treatment, Gap43 protein expression was significantly decreased (p<0.05). CONCLUSION: The results of this study showed that LIFU is an alternative treatment that can effectively relieve spastic reactions caused by SCI, possibly by reducing abnormal neuroplasticity or axon regeneration below the SCI segment.