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

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.


Introduction
Neuropathic pain (NP) is defined as pain originating from primary lesions and dysfunction of the somatosensory system, either at the peripheral or central level [1]. Many studies have been conducted on this type of pain, and some progress has been made, but many challenges remain in the clinical treatment of NP [2]. The main clinical manifestations include spontaneous pain, persistent (or paroxysmal) pain, induced pain, paresthesia, numbness, and tingling [2,3]. As a refractory and chronic pain that can manifest in various ways, including as chronic low back pain or sciatica, NP is a severe problem. It affects 6.9%-10% of the population worldwide and seriously diminishes patient's quality of life [2], increasing the economic burden on the patient's family and on society [4][5][6].
The etiology and mechanism of NP are complicated and unclear. Recently, an increasing amount of evidence has shown that downregulation of potassium chloride cotransporter 2 (KCC 2 ) in the spinal cord plays an important role in NP. KCC 2 is an ion transporter protein present in mature neurons of the central nervous system (CNS) and can remove Cl − from the cytoplasm to the extracellular space [7]. After peripheral nerve injury (PNI), the expression of KCC 2 on the neuronal membrane is downregulated, and the concentration of Cl − ([Cl − ] i ) in nerve cells is upregulated, thereby reducing the inhibitory effect of the neurotransmitter γ-aminobutyric acid (GABA) [8][9][10]. GABA, the main inhibitory neurotransmitter in the mature CNS [11], can bind to the GABA receptor (GABA-R) to promote depolarization of the postsynaptic nerve membrane and mediate hyperpolarization and activity of the neuron [12]. Dysfunction of GABA-R eventually reduces inhibition of the spinal cord, leading to hyperexcitability of primary afferent neurons and activation by low-threshold mechanical sensory input. Simultaneously, the primary afferent neurons respond only to highthreshold (nociceptive) inputs under normal circumstances, thereby causing mechanical allodynia and NP [13,14]. NP has been successfully induced in rats by injecting microribonucleic acid (miRNA), which interferes with the transcription of KCC 2 , or a KCC 2 inhibitor [15,16]. All results suggest that downregulation of KCC 2 after PNI plays an essential role in the development of NP [17]. Many studies have found that increasing the expression of KCC 2 significantly relieves NP behavior [9,[18][19][20]. Therefore, learning how to increase the expression of KCC 2 following PNI has great potential value for treating NP.
After PNI, nociceptive stimulation leads to downregulation of the KCC 2 expression through a series of intracellular cascades in the brain-derived neurotrophic factor-(BDNF-) tropomyosin receptor kinase B (TrkB) pathway [21,22]. Calcium/calmodulin-dependent protein kinase type IV (CaM-KIV), phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB), and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) play essential roles in activation of the BDNF-TrkB pathway cascades and downregulation of the KCC 2 expression [10,23,24]. Rivera confirmed in transgenic mice that activation of the TrkB receptor by BDNF further inhibited the expression of KCC 2 at the transcriptional level via the intracellular phosphoinositide phospholipase C gamma ðPLCγÞ ⟶ Ca 2+ ⟶ CaMKIV ⟶ p − CREB cascade [23]. Recently, studies also have found that nociception such as PNI or inflammation can activate the ERK-mitogen-activated protein kinase (MAPK) pathway in the spinal dorsal horn, upregulate the intracellular p-ERK1/2 expression via a Ras⟶p-ERK1/2 cascade reaction, and inhibit the expression of KCC 2 at the transcriptional level, ultimately leading to NP [23,25,26]. Therefore, the CaMKIV-KCC 2 or p-ERK1/2-KCC 2 pathway plays a vital role in NP pathogenesis after PNI (Figure 1). At present, due to its complicated mechanism, there is still no satisfactory treatment for NP [27,28]. At the clinical level, conventional painkillers such as tricyclic antidepressants, anticonvulsants, nonsteroidal anti-inflammatory drugs (NSAIDs), antiepileptic drugs, and weak and strong opioids are often used for symptomatic relief, but with poor efficacy and many side effects [28][29][30]. Therefore, finding a suitable rehabilitation method for NP would have great clinical significance. As a form of noninvasive neuromodulation, low-intensity focused ultrasound (LIFU) has been confirmed safe for modulating brain activity in patients and animals with seizures [31], Alzheimer's disease and dementia [32], traumatic brain injury (TBI) [33], and depression [34]. LIFU's neuromodulatory mechanism includes mechanical, thermal, and cavitation effects [35]. The mechanical effect of LIFU plays an important role in neuromodulation, and its mechanism might be that acoustic radiation forces the bimolecular structure of the cell membrane to stretch through mechanical vibration, thereby interfering with the mechanically sensitive ion channels on the cell membrane and producing the corresponding biological effect [36,37].
Interestingly, King et al. applied LIFU to the CNS in epileptic rats and found that it could inhibit abnormal epileptic discharge by activating GABAergic neurons in the CNS [38].
However, whether spinal cord stimulation with LIFU can enhance the inhibitory effect and alleviate NP is still unclear. In this study, we loosely ligated the right tibial nerve and common peroneal nerve in rats to create a PNI model. After LIFU stimulation of the L4-L5 spinal cord section, we used the 50% paw withdraw threshold (PWT 50 ) to evaluate the rats' mechanical stimulation threshold; WB and IF were used to detect the expression changes of p-ERK1/2, CaMKIV, p-CREB, and KCC 2 in the lumbar spinal cord.

Materials and Methods
2.1. Animals. We acquired a total of 40 healthy male Sprague-Dawley (SD) rats (weight, 220-300 g) from Kunming Laboratory Animal Center (Kunming, China) for use in the experiment. All rats were housed at 25°C ± 2°C on a 12 h reverse light/dark cycle in separate cages (5 rats per cage) and had free access to food and water. All animal protocols were approved by the Animal Ethics Committee of Kunming Medical University (No. KMMU2020352).

Grouping and Experimental
Design. After 1 week of adaptation, all rats were randomly divided into four groups (10 per group): normal group, rats that received neither surgery nor treatment; sham group, rats in which nerves were exposed according to the PNI surgical method but not ligated; and LIFU − group and LIFU + group, rats that received PNI surgery and LIFU stimulation in parallel, except that the ultrasound (US) amplifier was always turned off during treatment in the LIFU − group.

PNI Model of NP.
We developed the PNI model using the selective nerve injury (SNI) method in strict accordance with the literature [39]. Rats were anesthetized by intraperitoneal (i.p.) injection of 1% sodium pentobarbital (40 mg/kg). We shaved the fur at the right knee joint's proximal end and made a 1 cm incision. The muscle was separated bluntly, layer by layer, followed by exposure of the three branches of the right sciatic nerve: the tibial nerve, the common peroneal nerve, and the sural nerve. The common peroneal and tibial nerves were loosely ligated with 4-0 silk in three places at 1 mm intervals. We carefully performed manipulations during ligation to avoid injuring the sural nerve. The branches of the right sciatic nerve were exposed but not ligated in sham group rats.
2.4. LIFU Stimulation of the L4-L5 Spinal Cord Section. LIFU stimulation was started on the third day after PNI surgery during the time range of 09 : 00-15 : 00 (Figure 2(a)). After administering mild mixed anesthesia with isoflurane and sodium pentobarbital, we fixed the rats on a table and applied a depilatory cream to remove the fur on their backs, exposing the L4-L5 spinal segment. The transducer was fixed on this segment, and the skin was covered, and the transducer gaps filled with an ultrasonic coupling agent (Aquasonic; Parker Laboratories, Fairfield, NJ, USA) without bubbles. Parameters were as follows: sine pulse wave frequency, 4 MHz; duty  Mechanical allodynia (PWT 50 ) 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, PWT 50 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.
3 Neural Plasticity solution for 5 min, dipping in 1% acid ethanol five times, staining with eosin solution for 2 min, dipping in graded alcohol (from a high to a low concentration) for 5 min per grade, washing in water for 15 min, dehydration with graded (from a low to a high concentration) alcohol, clearing with xylene, and mounting in resin. We used a digital microscope to observe the results of H&E staining.
2.7. Measurement of Mechanical Allodynia. Behavioral tests were performed in a controlled environment by investigators who were blinded to animal treatments. Each rat was separately placed in a metallic mesh cage (20 × 20 × 15 cm 3 ) and allowed to adapt to the environment for 20 min before the test. We used the up-and-down method to test PWT 50 as described in the literature [40]. A series of von Frey (VF) filaments (Stoelting, Wood Dale, IL, USA) with ascending degrees of stiffness (1.4, 2.0, 4, 6, 8, 10, 15, and 26 g) were used to irritate the ipsilateral plantar surface of the PNI paw. The first VF filament to be used was the 6 g filament, and appropriate force was used to bend each filament for 5 s. Licking, lifting, or removing the paw was considered a positive reaction. According to the negative or positive response, we applied a filament at a greater or lower degree of force. PWT 50 was calculated as follows: 50%g threshold = 10 xf +kδ ½ /10, 000: The PWT 50 test was performed presurgery for 1 day, and pre-LIFU stimulation was performed 1 day/week during the LIFU stimulation period.
2.8. Western Blotting (WB) Analysis. The spinal cord tissue (0.1 g) was dissected, homogenized via US, lysed with radioimmunoprecipitation assay (RIPA) buffer (RIPA: phenylmethylsulfonyl fluoride ½PMSF = 1 ml : 10 μl) on ice for 30 min, and centrifuged at 12,000 r/min for 30 min at 4°C; then, we harvested the supernatants. Total protein concentration was quantified via a bicinchoninic acid (BCA) assay kit (Biomed, Beijing, China), and all samples were equalized to 30 μg/10 μl. Samples (total protein, 30 μg) were resolved by 6%, 10%, and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore-Sigma, Burlington, MA, USA). We blocked the membranes with 5% fat-free milk at room temperature (RT) for 2 h and then incubated them overnight with primary antibodies at 4°C with gentle shaking. These antibodies included monoclonal antibodies (mAbs) against CaMKIV 2.10. Statistical Analyses. Data are presented as mean ± standard deviation (SD). We used SPSS version 23.0 (IBM Corp., Armonk, NY, USA) for all statistical analyses. Graph-Pad Prism software version 8.0 (GraphPad Software, Inc., San Diego, CA, USA) was used to generate graphs. After verifying that all data were normally distributed, we used oneway analysis of variance (ANOVA) to analyze PWT 50 , WB, and IF data. P < 0:05 was considered statistically significant.

H&E Staining of the L4-L5 Spinal Cord Section Was Used
to Observe the Safety of LIFU Stimulation. We saw no swelling or nuclear fragmentation of neurons, neutrophil infiltration, or bleeding under cross-sectional magnification (Figure 3(a), ×40; Figure 3(b), ×100) of this spinal cord section.

LIFU Stimulation
Increased the KCC 2 Expression in the L4-L5 Spinal Cord Section. After 4 weeks of LIFU stimulation, rats were sacrificed, and the L4-L5 spinal cord section was harvested for WB (Figure 4(a)) and IF (Figure 5(a)) 4 Neural Plasticity analyses. The results showed that the expression of the KCC 2 protein in rats in the LIFU + group was upregulated compared with those in the LIFU − group (P < 0:05). There was no difference between the normal and sham groups (P > 0:05; Figures 4(b) and 5(b)).

LIFU Stimulation Reduced the Expression of CaMKIV and p-CREB but Not of p-ERK1/2 in the L4-L5 Spinal Cord
Section of PNI Rats. PNI activates the MAPK pathway and leads to high expression of CaMKIV, p-ERK, and p-CREB [41]. In this study, WB (Figures 4(c)-4(h)) showed that the expression of CaMKIV, p-ERK1/2, and p-CREB increased in the LIFU − group. After 4 consecutive weeks of LIFU treatment, the expression of CaMKIV and p-CREB decreased compared with the LIFU − group (P < 0:05; Figures 4(f) and 4(h)). IF also showed that the expression of p-CREB decreased after LIFU stimulation for 4 weeks compared with the LIFU − group (P < 0:05; Figure 5(d)). Interestingly, there was no statistical difference in the p-ERK1/2 expression between the LIFU − and LIFU + groups, nor any significant difference in CaMKIV, p-ERK1/2, or p-CREB expression between the normal and sham groups (P > 0:05; Figures 4(d), 4(f), and 4(h)).

Discussion
Potassium chloride (K + -Cl − ) cotransporter 2 (KCC 2 ) is the only cationic chloride cotransporter expressed in mammalian neurons. It plays a prominent role in maintaining low [Cl − ] i , which is necessary for the function of GABA A and glycine receptors (GlyRs) and for mediating spinal cord inhibition [8,10]. After intrathecal application of KCC 2 inhibitor (2-[[(2S)-2-butyl-6,7-dichloro-2-cyclopentyl-1oxo-3H-inden-5-yl]oxy], or DIOA), heat-evoked withdrawal latency and innocuous brush stimulation are significantly reduced [16,42]. Our experimental data indicated that KCC 2 was downregulated in the PNI group, and PWT 50 was also lower in this group than in the normal and sham operation groups. All results showed that PNI led to downregulation of the KCC 2 expression, which weakens GABA A /GlyR-mediated inhibition and then leads to NP [43]. All of the above changes are important factors contrib-uting to the development and maintenance of NP. To further investigate the mechanism of NP, we found that enhancing the KCC 2 function pharmacologically restored spinal cord inhibition and reduced allodynia [9]. In our study, pain behavior improved (Figure 2(b)), and the KCC 2 expression was upregulated (Figure 4(b)) after 4 weeks of LIFU stimulation. Therefore, the expression of KCC 2 in the spinal cord played an important role in the pathogenesis of NP, and upregulation of the KCC 2 expression could potentially alleviate NP.
After PNI, the downregulation of KCC 2 is closely related to activation of the BDNF-TrkB pathway and intracellular cascade reactions mediated by CaMKIV, p-CREB, and p-ERK [21][22][23][24]. Intrathecal application of a TrkB blocker significantly improves downregulation of the KCC 2 expression on the membranes of spinal dorsal horn neurons induced by inflammatory pain [44]. In Kitayama's research, short interfering RNA (siRNA) was used to knock down zinc transporter-1 (ZnT-1), which led to inhibition of the BDNF-TrkB pathway, downregulation of p-CREB, upregulation of KCC 2 , and improvement of the withdrawal threshold [19]. After intrathecal injection of p-ERK blockers, chronic NP induced by oxaliplatin was also significantly alleviated in rats [45]. López-Alvarez and Li applied electroacupuncture to stimulate rats with chronic constriction injury (CCI) and found that it could effectively improve the KCC 2 expression, the mechanical withdrawal threshold, and thermal withdrawal latency [18,20]. Therefore, inhibition of the BDNF-TrkB pathway and cascade reactions mediated by CaMKIV, p-CREB, and p-ERK, as well as upregulation of the KCC 2 expression, could effectively alleviate NP. In this study, we stimulated the spinal cord with LIFU and confirmed the efficacy of LIFU in treating NP. To our knowledge, this study was the first to use LIFU to stimulate the spinal cord in order to regulate NP.
Moreover, we found that CaMKIV and p-CREB were downregulated (Figures 4(f 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. 6 Neural Plasticity and the behavior of pathological pain is relieved [7,46]. Therefore, we speculate that LIFU might alleviate pathological pain due to PNI by inhibiting CaMKIV and p-CREB expression and upregulating the KCC 2 expression in neurons. Interestingly, LIFU stimulation did not change the expression of p-ERK1/2 in the spinal cords of PNI rats (Figure 4(d)). While the exact underlying mechanism is unknown, there are several possible explanations. First, in NP rat models, the BDNF-TrkB pathway can activate CaM-KIV by increasing the concentration of Ca 2+ in neurons via the PLCγ-IP 3 pathway, whereas p-ERK is activated through the TrkB-Ras pathway [22,26]. Second, CaMKIV activation depends on the concentration of Ca 2+ in neurons. The mechanical forces of LIFU can affect voltage-gated calcium and sodium channels (VGCCs, VGSCs) in the plasma membrane [35,47], causing transient intracellular Ca 2+ concen-tration changes in various cells [48]. This mechanism can be used in treatments such as mesenchymal stem cell (MSC) homing [36], neuromodulation in the brain [47], or immunotherapy with tumor US [49]. Therefore, we propose that LIFU might affect CaMKIV activation by interfering with the transient concentration of Ca 2+ in neurons but without affecting the p-ERK1/2 expression. However, the specific mechanism of action remains unclear, requiring further research.

Neural Plasticity
In this study, we transected the spinal cord at L4-L5 and performed H&E staining to observe the safety of LIFU on the spinal cord. Our results showed no swelling, nuclear fragmentation of neurons, neutrophil infiltration, or bleeding (Figures 3(a) and 3(b)). Therefore, these results indicated that LIFU was a safe method for treating the spinal cord.
Overall, our study demonstrated that (i) LIFU stimulation of the spinal cord could effectively improve neuropathic pain behavior induced by peripheral nerve injury, which has potential value in the clinical treatment of NP; (ii) LIFU stimulation of the spinal cord might affect the expression of CaM-KIV, CREB, and KCC 2 ; and (iii) stimulation of the spinal cord with LIFU was safe.

Limitations
Our study had some limitations. First, we established only a short treatment period and did not evaluate the long-term efficacy of US therapy. Second, we selected only one time point at which to measure the expression of CaMKIV, p-CREB, p-ERK, and KCC 2 . Third, we found that LIFU could affect the expression of CaMKIV, p-CREB, and KCC 2 , but we failed to explore the specific mechanism by which it affected the expression of the above proteins. Thus, further experiments are needed.

Conclusions
We found that LIFU could effectively alleviate NP in rats with PNI by increasing the expression of KCC 2 in the spinal dorsal corner. Moreover, LIFU upregulated the expression of KCC 2 , possibly by inhibiting activation of the CaMKIV-KCC 2 pathway.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.