Electroacupuncture at Dazhui (GV14) and Mingmen (GV4) acupoints promoting nerve repair in rat model of spinal cord injury: a study based on diffusion tensor imaging

Introduction

Spinal cord injury (SCI) is a highly debilitating condition affecting the central nervous system (CNS). It can cause severe motor and sensory dysfunction in the limbs below the injury site, along with complications such as bladder dysfunction and pressure sores (1). According to statistics (2), there are approximately 10.4 to 83 cases of SCI patients per million people in high-income countries, and 12.1 to 57.8 cases in low-income countries annually. Importantly, the absolute burden of SCI is rising, with projections indicating that global cases will exceed 14.5 million by 2050 (3,4). Currently, there is no effective clinical treatment for SCI. Consequently, promoting neural repair following SCI has become an urgent medical issue that requires resolution.

Following SCI, the nerve fibers in the affected area experience varying degrees of rupture, which leads to difficulties in nerve impulse conduction and results in the loss of motor, sensory, and autonomic functions below the level of injury (5). Therefore, promoting the regeneration and repair of damaged nerves is of great significance for the transmission of nerve signals and the recovery of motor function after SCI. Previous studies have demonstrated that nerve growth guidance cues are involved in the neural repair after SCI (6). Netrins are a kind of laminin-related secreted proteins that influence axon guidance and cell migration during neural development (7). Among the netrin family, netrin-1 acts as a nerve growth guidance cue involved in the injury and repair processes of the CNS (8,9). Netrin-1 has been confirmed to play a dual role in axon growth, mediating both attraction and repulsion signals by binding to different receptors (10,11). The netrin1-mediated axon attraction signaling pathway involves several key molecules, including netrin-1, deleted in colorectal cancer (DCC), and ras-related C3 botulinum toxin substrate 1 (Rac1). Upon binding to its receptor DCC, netrin-1 initiates an attractive guidance signal that precisely modulates Rac1 activity, subsequently inducing the formation of both filopodia and lamellipodia within the growth cone (12-14). This molecular mechanism regulates the targeted growth of axons, ultimately ensuring the accurate guidance of growth cone extension. Therefore, regulating the axon attraction signal pathway mediated by netrin-1 is crucial for promoting effective regeneration and repair of nerve axons.

Electroacupuncture (EA) is a treatment method that integrates traditional acupuncture with micro-pulse currents. This method is characterized by its low cost, established efficacy, high safety, and minimal side effects. Clinical and animal experimental evidence indicate that EA not only enhances the motor function following SCI but also effectively alleviates neuropathic pain, neurogenic intestinal dysfunction, and neurogenic bladder dysfunction resulting from such injuries (15-19). Our previous studies have confirmed that EA at “Dazhui” (GV14) and “Mingmen” (GV4) acupoints could significantly relieve the pathological changes, improve neurological function, and promote nerve regeneration in rats with SCI (20-23). However, the underlying mechanisms remain to be further investigated.

Diffusion tensor imaging (DTI) is a specialized magnetic resonance imaging (MRI) technique that facilitates the in vivo examination of the white matter fiber structure within the CNS. It can visually display the changes in fiber connections in the living spinal cord through diffusion tensor tractography (DTT) (24). In this research, we implemented EA with different treatment cycles (7, 14 and 28 days) at GV14 and GV4 acupoints in SCI model rats. DTI was employed to observe the changes of nerve fiber bundle structure in SCI model rats after EA treatment, aiming to evaluate the effect of EA on the repair and regeneration of injured nerves. Furthermore, we investigated the effects of EA on the expression of netrin-1, DCC, Rac1 and F-actin in the spinal cord of SCI model rats to evaluate its regulatory role in axon guidance signaling. This investigation further elucidated the intrinsic mechanisms by which EA promotes nerve regeneration and repair in rats with SCI. We present this article in accordance with the ARRIVE reporting checklist (available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1816/rc).

Methods

Experimental animals

One hundred and forty-four male adult Sprague Dawley rats, weighing 220.0±20.0 g were obtained from Sibeifu (Beijing) Biology Technology Co., Ltd. [Animal Lot: SCXK(Jing) 2019-0010]. The animals were housed in the Experimental Animal Center of the Beijing University of Chinese Medicine at a controlled temperature (24±2 ℃) and under a 12-h dark/light cycle, with sterile drinking water and a standard pellet diet available ad libitum. All rats were acclimatized to the environment for 7 days before experimentation, and all experimental procedures were performed under a project license (No. BUCM-2024042505-2065) granted by the Animal Ethics Committee of Beijing University of Chinese Medicine, in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

All rats were randomly divided into four groups (n=36/group): the Normal group, Sham group, SCI group, and EA group. Rats from the above four groups were randomly assigned into the following subgroups: 7- (n=12), 14- (n=12), and 28-day (n=12). The experimental flow diagram is shown in Figure 1A,1B.

Figure 1 Schematic diagram of the experimental design and electroacupuncture treatment. (A) Experimental animal grouping. (B) Time axis diagram of experimental design. (C) The schematic diagram of EA treatment. Created in BioRender. Yang Y. (2026) https://BioRender.com/2caw7jp. DTI, diffusion tensor imaging; IF, immunofluorescence; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; SCI, spinal cord injury; SD, Sprague Dawley; WB, Western blot.

Establishment of SCI model

The SCI model was constructed using a modification of Allen’s method (25). Briefly, anesthesia was induced using 4% isoflurane in oxygen (1 L/min) and maintained with 2% isoflurane in oxygen (1 L/min) for the rats. A 2-cm longitudinal incision was made in the posterior midline, and the muscles were separated layer by layer to expose the T9-T11 spine. The lamina at T10 was removed to expose the spinal cord. Following this, the Infinite Horizon Impactor (IH-0400, Lexington, KY, USA) injury device was employed to accurately impact the exposed dura mater (200 kilodyne) to induce a moderate contusion. The criteria for a successful SCI model include: spasmodic tail swing, congestion or hematoma in the dura mater, and Basso-Beattie-Bresnahan (BBB) score of 0 within 24 hours after operation. Subjects meeting these criteria were included in the experiment. After covering the dura mater with a gelatin sponge, the wound was sutured layer by layer. The penicillin sodium (20,000 U; CP8271-5g, Coolaber) was administered to rats through intramuscular injection once a day for 3 days after surgery to prevent infection, and bladder massage was performed 2–3 times daily to assist with urination.

Rats in the Sham group underwent the same surgical procedure, including lamina removal, but the spinal cord was not damaged.

EA treatment

In the EA group, the rats were treated with EA 24 h after successful modeling. According to our previous research (21,23,26), Dazhui (GV14) and Mingmen (GV4) acupoints were selected for EA treatment. Specifically, the disposable sterile acupuncture needles (diameter, 0.30 mm; length, 25 mm; Zhongyan Taihe, Beijing, China) were inserted into GV14 and GV4 points at an angle of 15–45 degrees, with a depth of 0.5–0.7 cm. Needles were connected to the EA therapeutic apparatus (SDZ-II, Beijing Huatuo Industrial Development Co., Ltd., Beijing, China) at a frequency of 2 Hz and an intensity of 1 mA for 20 min. The schematic diagram of the EA treatment protocol is shown in Figure 1C. According to the three subgroups, the intervention duration was divided into 7, 14, or 28 days.

To minimize any discrepancies due to stress, rats in the Normal, Sham and SCI groups received the same restriction as those in the EA group, once daily for 7, 14, or 28 days.

BBB scale score

The hindlimb motor function of rats was evaluated by BBB score on the 1st, 7th, 14th, 21st and 28th day after SCI modeling according to previous method (27). Rats were placed on the open flat ground and allowed to crawl for 5 min. Their hindlimb movements were observed and recorded by three experienced researchers who were not involved in the treatment. Scores were assigned on a scale from 0 (indicating no observed hindlimb movements) to 21 (indicating normal gait), based on criteria including limb movement, gait, coordination, and paw placement.

Inclined plane test

The hindlimb motor function of rats was evaluated by inclined plate test on the 1st, 7th, 14th, 21st and 28th day after SCI modeling. Rats were placed on a 30° inclined plate and allowed to adapt for 1min, and then the angle of inclined plate was gradually increased, with 5° as a unit. If the rats keep 5 s on the inclined plate with a certain angle, continue to increase the angle until the rats can’t maintain their position for 5 s without sliding, and conduct the same experimental operation four times at this angle. Among the five experimental operations, If the rats remained for 5 s fewer than three times, it will be recorded as the maximum angle; if the rats keep 5 s for more than three times (including three times), the angle was further increased until the maximum angle was determined.

MRI and DTI

The rats were examined by MRI and DTI using a uMR 9.4T (United Imaging Life Science Instrument, Wuhan, China) on the 1st, 7th, 14th, and 28th day after SCI modeling. During the imaging sessions, anesthesia was induced with 4% isoflurane in oxygen (1 L/min) and maintained at 2.5–3% isoflurane in oxygen (1 L/min) for the rats. Anesthetized rats were positioned prone at the center of the magnetic field, with their body temperature regulated to normal levels, and their respiratory frequency closely monitored. T2-weighted imaging (T2WI) was initially acquired using a spin-echo sequence with the following parameters: TR (repetition time) =3,000 ms, TE (echo time) =54.08 ms, TA (acquisition time) =4 min 27, MTX (matrix size) =176×176, SR (spatial resolution) =0.2×0.2×0.3 mm/pixel, 84 slices in total, and field of view (FOV) = 3.5 cm × 3.5 cm. DTI has TR =2,236 ms, TE =21.8 ms, TA =3 min 50 s, slice thickness =1 mm, FOV = 15 mm × 17 mm, matrix =74×84, and b value =800 s/mm².

Lesion areas were calculated via threshold-based segmentation of sagittal T2WI using ImageJ. The original DTI data were analyzed and processed by DSI Studio Software (Chen version 2022) after image acquisition. The regions of interest (ROIs) were delineated within 1 cm above and below the center of spinal cord lesion in rats, and the fractional anisotropy (FA), radial diffusivity (RD), and mean diffusivity (MD) were calculated. The average value of three measurements was taken as the final parameter value. The DTT was reconstructed with the focus area as the center.

Western blot (WB) analysis

The rats of 7- (n=6), 14- (n=6), and 28-day (n=6) subgroups from the Normal, Sham, SCI and EA groups were euthanized with intraperitoneal injection of pentobarbital sodium (200 mg/kg). The injured spinal cord segment from each rat was collected for analysis. Total protein from each spinal cord tissue sample was extracted using RIPA Lysis Buffer and the supernatant was collected. After determining the protein concentration, the protein was denatured and the SDS-PAGE gel was performed. Following electrophoresis, the proteins were transferred from the gel to a 0.45 µm PVDF membrane. The membranes were then blocked with 5% skimmed milk at room temperature for 30 min. The rabbit polyclonal antibody to netrin-1 (ANR-121-50UL, Invitrogen, California, USA) and β-actin (20536-1-AP, Proteintech, Wuhan, China) were added separately and incubated at 4 ℃. After washing, the secondary antibodies (anti-rabbit, SA00001, Proteintech, Wuhan, China) were added. An ECL detection kit (P0018S, Beyotime, Shanghai, China) was then applied to the membrane. The bands were semi-quantified using a gel analyzing system, and the ratio of the gray value of the target protein to that of the internal reference protein was used as the relative expression.

Immunofluorescence (IF) staining

The rats of 7- (n=6), 14- (n=6), and 28-day (n=6) subgroups from the Normal, Sham, SCI and EA groups were injected with 3% sodium pentobarbital (2.0 ml/kg) anesthesia and perfused with paraformaldehyde. Approximately 1 cm of spinal cord tissue containing the lesion center segment from each rat was collected. After sucrose dehydration and embedding, the spinal cord tissues were sectioned by a Microtome Cryostat (CM1900, Leica Corporation, Wezlar, Germany). The rabbit polyclonal antibody to netrin-1 (1:200, ANR-121-50UL, Invitrogen, California, USA), the rabbit polyclonal antibody to DCC (1:200, 19123-1-AP, Proteintech, Wuhan, China) and the rabbit polyclonal antibody to F-actin (1:200, bs-1571R, Bioss, Beijing, China) were used as the primary antibodies. The corresponding secondary antibody was donkey anti-rabbit IgG Alexa Fluor 594 (1:200, ab150073, Abcam, Cambridge, USA). After washing and blocking, netrin-1 antibody was added dropwise to the sections and incubated overnight at 4 ℃. After washing, the sections were treated with secondary antibody. Finally, the sections were stained with Fluoroshield Mounting Medium with DAPI (ab104139, Abcam, Cambridge, USA). The expression of DCC and F-actin was examined using the same staining protocol. Identical image settings were used for confocal laser scanning microscope observations (FV3000, Olympus Corporation, Tokyo, Japan). The mean optical densities of netrin-1, DCC and F-actin were analyzed using ImageJ in each group.

Real-time quantitative polymerase chain reaction (RT-PCR)

The rats of 7- (n=6), 14- (n=6), and 28-day (n=6) subgroups from the Normal, Sham, SCI and EA groups were euthanized with intraperitoneal injection of pentobarbital sodium (200 mg/kg). The injured spinal cord segment from each rat was collected. The total RNA of spinal cord was extracted by Trizol reagent, and the concentration and purity were determined. Reverse transcription of RNA into cDNA was performed according to the instructions provided by the reverse transcription kit. PCR amplification was performed with netrin-1, Rac1, and DCC primers using cDNA as a template. GAPDH was selected as the endogenous reference gene. The netrin-1 forward primer sequence was 5'-CCTCACCGACCTCAACAATCC-3', whereas the reverse primer sequence was 5'-GCGGCACTGAGTGGAATAGAAT-3'. The Rac1 forward primer sequence was 5'-CCTGCCTGCTCATCAGTTACAC-3', whereas the reverse primer sequence was 5'-GGGACGCAGTCTGTCATAATCT-3'. The DCC forward primer sequence was 5'-CAAGAACAGGAAATGAAGCCGA-3', whereas the reverse primer sequence was 5'-CTCCCACCCAACAACGAATACTT-3'. The GAPDH forward primer sequence was 5'-CTGGAGAAACCTGCCAAGTATG-3', and its reverse primer sequence was 5'-GGTGGAAGAATGGGAGTTGCT-3'. The reaction conditions of reverse transcription were 25 ℃ for 5 min, 42 ℃ for 30 min, 85 ℃ for 5s and 4 ℃. Amplification conditions: pre-denaturation at 95 ℃ for 30 s; denaturing at 95 ℃ for 15s, annealing/stretching at 60 ℃ for 30 s, a total of 40 cycles. The melting curve was set to 95 ℃ for 5 s, 65 ℃ for 1 min and 97 ℃ for 30 s. The relative expression of mRNA was calculated using the equation 2^−ΔΔCT.

Statistical analysis

The statistical analysis was performed by SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA). Accordingly, the required graphs were generated by GraphPad Prism 8 software. All data were expressed as the mean ± standard deviation. BBB score, inclined plane test and DTI data were analyzed using repeated measures analysis of variance. In other experiments, one-way ANOVA with the least significant difference (LSD) method was used to compare the variability among groups when the data were normally distributed or had homogenous variance. Otherwise, the Dunnett’s T3 test would be used. Statistical significance was defined as P<0.05, while highly statistically significant as P<0.01.

Results

Effect of EA on motor function in rats with SCI

On the 1st, 7th, 14th, 21st and 28th days after SCI modeling, the BBB scores and the angle of the inclined plane of the SCI group were significantly lower than those of the Normal group and Sham group (P<0.01) (Figure 2). The BBB scores and the angle of the inclined plane of the EA group on the 7th, 14th, 21st and 28th days after SCI modeling were significantly higher than those of the SCI group (P<0.01) (Figure 2).

Figure 2 Effect of EA on motor function in rats with SCI. (A) The BBB scores of rats in each group (n=12). (B) Results of inclined plate test of rats in each group (n=12). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲▲, P<0.01 versus the SCI group. BBB, Basso-Beattie-Bresnahan; EA, electroacupuncture; SCI, spinal cord injury.

Effects of EA on morphology changes in rats with SCI

On the 1st, 7th, 14th, and 28th days after SCI modeling, rats in the Normal and Sham groups exhibited no obvious compression of the spinal cord. The spinal cord structure remained intact, and no abnormal signal was found. In the spinal cord of rats within both the SCI and EA groups, compression and deformation were observed in the injured area, leading to a disordered structure. Necrotic cavity and reactive hyperplastic tissue can be observed in the center of spinal cord lesion, and hyperintensity on T2WI showed inflammatory reaction, edema or local effusion. The lesion area of spinal cord tissue progressively alleviated as time went on. Compared with the SCI group, the EA group exhibited a more complete organizational structure and a smaller lesion area on the 7th, 14th, and 28th days after SCI modeling (P<0.01) (Figure 3). However, on the 1st day following the injury, there was no significant difference in the lesion area between the EA group and the SCI group.

Figure 3 Effects of EA on morphology changes in rats with SCI. (A) T2WI shows sagittal images of the spinal cord at 1, 7, 14, and 28 days post-injury of the same rat in each group. The white arrows indicate the lesion epicenter. (B) The lesion area at 1, 7, 14, and 28 days post-injury in each group (n=3). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲▲, P<0.01 versus the SCI group. EA, electroacupuncture; SCI, spinal cord injury; T2WI, T2-weighted imaging.

Effects of EA on nerve fiber bundles in rats with SCI assessed by DTI

DTT images showed greater homogeneity of spinal cord fiber bundles in both the Normal and Sham groups, with blue DTI signals uniformly filling the entire spinal cord structure, and the fiber bundles were structurally intact with a continuous alignment. In both the SCI group and the EA group, nerve fiber bundles were fractured in the injured area of the spinal cord, the integrity of which was damaged, and the consistency was reduced. In the EA group, the injury to the spinal cord fiber bundles was significantly alleviated compared to the SCI group, with clear evidence of broken fiber bundle (Figure 4).

Figure 4 Effects of EA on nerve fiber bundles in rats with SCI. DTT images of the spinal cord at 1, 7, 14, and 28 days post-injury of the same rat in each group. Representative DTT reconstructions showing the integrity and continuity of spinal cord white matter tracts at the lesion site and adjacent segments. Different colors indicate the main direction of water molecule diffusion. The movement of water molecules along the rostro-caudal direction of the spinal cord fiber bundles shows a uniform blue color. Other colors indicate that water molecules diffuse in other directions due to the rupture of fiber bundles. P indicates posterior. EA, electroacupuncture; DTT, diffusion tensor tractography; SCI, spinal cord injury.

On the 1st, 7th, 14th, and 28th days after SCI modeling, the FA value at the center of the spinal cord lesion in both the SCI and EA groups were significantly lower than that in the Normal and Sham groups (P<0.01), and the MD and RD values were observably higher than those in the Sham and Normal groups (P<0.01). Compared with the SCI group, the FA value at the center of the spinal cord lesion in the EA group increased notably on the 7th, 14th and 28th day after SCI modeling (P<0.01 or P<0.05), while the MD and RD values decreased strikingly (P<0.01 or P<0.05) (Figure 5A-5C). On the 1st day after SCI modeling, there were no significant differences in FA, MD and RD values between the EA group and the SCI group.

Figure 5 Effects of EA on nerve fiber bundles in rats with SCI. (A) The FA value of the center of spinal cord lesion at 1, 7, 14, and 28 days

post-injury in each group (n=3). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲, P<0.05; ^▲▲, P<0.01 versus the SCI group. (B) The MD value of the center of spinal cord lesion at 1, 7, 14, and 28 days post-injury in each group (n=3). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲▲, P<0.01 versus the SCI group. (C) The RD value of the center of spinal cord lesion at 1, 7, 14, and 28 days post-injury in each group (n=3). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲, P<0.05; ^▲▲, P<0.01 versus the SCI group. (D) Spearman correlation analysis between DTI parameters and BBB scores at 7 days post-injury. (E) Spearman correlation analysis between DTI parameters and BBB scores at 14 days post-injury. (F) Spearman correlation analysis between DTI parameters and BBB scores at 28 days post-injury. BBB, Basso-Beattie-Bresnahan; DTI, diffusion tensor imaging; EA, electroacupuncture; FA, fractional anisotropy; MD, mean diffusivity; RD, radial diffusivity; SCI, spinal cord injury.

Correlation analysis between DTI parameters and BBB scores

Spearman correlation analysis showed that after 7, 14, and 28 days of injury, the FA value in the center of spinal cord lesion in rats was positively correlated with BBB scores (day 7, r=0.885, P<0.01; day 14, r=0.936, P<0.01; day 28, r=0.894, P<0.01), while the MD and RD values were negatively correlated with BBB scores (MD, day 7, r=−0.913, P<0.01; day 14, r=−0.922, P<0.01; day 28, r=−0.914, P<0.01; RD, day 7, r=−0.880, P<0.01; day 14, r=−0.929, P<0.01; day 28, r=−0.601, P<0.05) (Figure 5D-5F).

Effects of EA on nerve growth guidance cues netrin-1 and its receptor DCC in rats with SCI

The results of IF, WB and RT-PCR showed that compared with the Normal and Sham groups, the mean optical density, relative expression, and mRNA expression of netrin-1 in the spinal cord of the SCI group decreased significantly on the 7th, 14th, and 28th days after SCI modeling (P<0.01). The mean optical density, relative expression, and mRNA expression of netrin-1 in the spinal cord of the EA group were markedly higher than those observed in the SCI group on the 7th, 14th, and 28th days after SCI modeling (P<0.01, Figure 6).

Figure 6 Effects of EA on nerve growth guidance cues netrin-1 in rats with SCI. (A) Representative images of IF staining of netrin-1 in each group, magnification ×400, Scale bars: 80 μm. (B) The mean optical density of netrin-1 in each group (n=6). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲▲, P<0.01 versus the SCI group. (C) The mRNA expression of netrin-1 in each group (n=6). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲▲, P<0.01 versus the SCI group. (D) Typical protein bands of netrin-1 in each group. (E) The relative expression of netrin-1 in each group (n=6). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲▲, P<0.01 versus the SCI group. EA, electroacupuncture; IF, immunofluorescence; SCI, spinal cord injury.

Compared with the Normal and Sham groups, the mean optical density and mRNA expression of DCC in the spinal cord of the SCI group decreased significantly on the 7th, 14th, and 28th days after SCI modeling (P<0.01). The mean optical density and mRNA expression of DCC in the spinal cord of the EA group were markedly higher than those observed in the SCI group on the 7th, 14th, and 28th days after SCI modeling (P<0.01) (Figure 7A-7C).

Figure 7 Effects of EA on the expression of DCC and Rho GTPase Rac1 in rats with SCI. (A) Representative images of IF staining of DCC in each group, magnification ×400, Scale bars: 80 μm. (B) The mean optical density of netrin-1 in each group (n=6). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲▲, P<0.01 versus the SCI group. (C) The mRNA expression of DCC in each group (n=6). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲▲, P<0.01 versus the SCI group. (D) The mRNA expression of Rac1 in each group (n=6). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲▲, P<0.01 versus the SCI group. DCC, deleted in colorectal cancer; EA, electroacupuncture; IF, immunofluorescence; Rac1, ras-related C3 botulinum toxin substrate 1; SCI, spinal cord injury.

Effects of EA on the expression of Rho GTPase Rac1 in rats with SCI

Compared with the Normal and Sham groups, the expression of Rac1 mRNA in the spinal cord of the SCI group decreased significantly on the 7th, 14th, and 28th days after SCI modeling (P<0.01). The expression of Rac1 mRNA in the spinal cord of the EA group was notably higher than that observed in the SCI group on the 7th, 14th and 28th days (P<0.01) (Figure 7D).

Effects of EA on cytoskeleton protein expression in rats with SCI

Compared with the Normal group and Sham group, the mean optical density of F-actin in the spinal cord of the SCI group decreased significantly on the 7th, 14th, and 28th days after SCI modeling (P<0.01). The mean optical density of F-actin in the spinal cord of the EA group was notably higher than that in the SCI group on the 7th, 14th and 28th days (P<0.01) (Figure 8A,8B).

Figure 8 Effects of EA on cytoskeleton protein expression in rats with SCI. (A) Representative images of IF staining of F-actin in each group, magnification ×400, Scale bars: 80 μm. (B) The mean optical density of F-actin in each group (n=6). **, P<0.01 versus the Normal group; ^##, P<0.01 versus the Sham group; ^▲▲, P<0.01 versus the SCI group. (C) Spearman correlation analysis between Rac1 mRNA expression and F-actin fluorescence intensity at 7 days post-injury. (D) Spearman correlation analysis between Rac1 mRNA expression and F-actin fluorescence intensity at 14 days post-injury. (E) Spearman correlation analysis between Rac1 mRNA expression and F-actin fluorescence intensity at 28 days post-injury. EA, electroacupuncture; IF, immunofluorescence; Rac1, ras-related C3 botulinum toxin substrate 1; SCI, spinal cord injury.

Spearman correlation analysis showed that after 7, 14, and 28 days of injury, the Rac1 mRNA expression was positively correlated with F-actin fluorescence intensity (day 7, r=0.870, P<0.0001; day 14, r=0.901, P<0.0001; day 28, r=0.864, P<0.0001) (Figure 8C-8E).

Discussion

The continuous disruption of spinal nerve fibers caused by SCI is an essential factor leading to a range of neurological dysfunctions (28). The growth of neuronal axons is a critical process for nerve repair and regeneration. Studies have found that axon regeneration relies on the intricate interplay of various molecules within the netrin-1-mediated attractive signaling pathway, which synergistically promotes the repair of nerve fibers (29). Specifically, when netrin-1 binds to its receptor DCC, it mediates an attractive signal for axons and conveys axon guidance information to the Rho GTPase Rac1. Rac1 subsequently enhances axon growth and stabilization by regulating the actin cytoskeleton (14). In this study, we observed that EA at GV4 and GV14 significantly improved the motor function of SCI model rats, regulated the expressions of netrin-1, DCC and Rac1, and promoted the regeneration and repair of nerve fibers. We speculated that the mechanism by which EA promotes nerve repair in SCI model rats may be associated with the axon attractive signaling pathway mediated by netrin-1.

The Governor Vessel traverses the midline of the human back and is closely related to the spinal cord. Traditional Chinese medicine holds that injury to the Governor Vessel is a primary cause of SCI. GV14 and GV4 are major acupoints on the Governor Vessel, traditionally considered to nourish the kidneys and generate the marrow. Our previous studies have confirmed the efficacy of EA at GV14 and GV4 in treating SCI (20-23), yet the underlying mechanisms remain to be further explored. Therefore, we continue to select these two acupoints for EA intervention in the present study, aiming to elucidate the mechanisms by which EA promotes neural repair.

Motor dysfunction is the main symptom of SCI. The BBB score and the inclined plate test are behavioral methods used to assess the motor function of hind limbs in rats. The results showed that on the first day after the surgery, the BBB scores of rats in both the SCI and EA groups were 0, which was significantly different from those in the Normal and Sham groups, thereby confirming the successful establishment of the SCI model. Furthermore, the behavioral test results demonstrated significant differences in the EA group compared to the model group starting from the 7th day, with these differences persisting until the 28th day. This indicates that EA can enhance the motor function of rats with SCI, and its therapeutic effects become increasingly pronounced with the extension of the treatment duration, which is consistent with our previous research results (21,22,26).

DTI can evaluate the integrity and function of spinal cord nerve fiber bundles by measuring the diffusion of water molecules in tissues (30), making it an ideal tool for assessing the therapeutic effects following SCI treatment. FA, MD and RD are the basic parameters of DTI, which can sensitively reflect the changes in spinal nerve repair (24,31). FA indicates the integrity and conduction function of nerve fiber bundles, quantitatively expressing the pathophysiological state of these bundles (32). RD is considered a potential marker for axonal and myelin injury (33). MD reflects the overall diffusion levels and resistance of water molecules, and its increase is related to demyelination of nerve fibers or axonal loss (34). The results of the Spearman correlation analysis in this study showed a significant correlation between BBB scores and the FA, MD, and RD values at the center of spinal cord lesions in rats. This finding aligns with previous research (35-37), suggesting that DTI parameters can evaluate the therapeutic effects of EA on rats with SCI to a certain extent across different treatment courses. In this study, we found that the FA value in the SCI group was notably lower than that in both the Sham and Normal groups, while the MD and RD values were prominently higher. Cell necrosis and demyelination were observed at the center of the spinal cord lesion after SCI, resulting in the disruption of nerve fiber integrity. This disruption reduces the anisotropy of water molecule diffusion, consequently leading to a decrease in the FA value (38). After demyelination of spinal cord nerve fibers, the resistance to the diffusion of water molecules in the direction perpendicular to the nerve fibers decreases, which may be the main reason for the increase of MD and RD values after SCI (39,40). EA significantly improved the FA, MD, and RD values at the center of spinal cord lesions in rats. It alleviated damage to the spinal cord fiber tracts and facilitated the repair and reconstruction of the injured fibers. Importantly, T2WI and DTI analysis performed 24 h
post-injury, prior to the commencement of treatment, confirmed no significant differences in the initial lesion area or DTI parameters between the EA and SCI groups. This finding ensures that the observed long-term benefits in the EA group can be attributed to the intervention rather than disparities in the initial severity of the injury.

The primary reason for the failure of nerve repair after SCI is the inability of regenerated axons to establish precise connections with target tissues. Researchers believe that the accurate guiding function of nerve growth guidance cues may be a critical factor influencing axon regeneration (41). Netrins are attractive nerve growth guidance cues (42,43), widely expressed in the white matter, gray matter neurons, and oligodendrocytes of the mammalian spinal cord, where they play a regulatory role in axon regeneration (44). DCC is an attractive receptor for netrin-1 (45), which is essential for axon growth and the formation of neural connections (12). The interaction between DCC and netrin-1 accurately modulates the growth direction of the axon growth cone, thereby regulating the targeted growth of nerve axons through its attractive properties (46). Studies have shown that the activation of the endogenous netrin-1/DCC pathway not only stimulates nerve axon regeneration but also facilitates functional recovery following neurological injury (47). The guidance cues transmitted by netrin-1/DCC initiate the intracellular signal transduction mechanism via influencing Rho GTPases, thereby promoting cytoskeleton reconstruction within the axon (48). Rac1 is a pivotal member of the Rho GTPase family, which is involved in regulating the actin cytoskeleton (49). Netrin-1 binds to the DCC receptor, inducing the formation of DCC homodimers. This activation of DCC further stimulates Rac1, promoting cytoskeletal reorganization and the formation of neurites, thereby assisting injured nerve cells in re-establishing connections (50,51). Therefore, the axon attraction signal pathway mediated by netrin-1 is crucial for regulating nerve repair and regeneration after SCI. In this experiment, we observed the expression levels of netrin-1, DCC and Rac1 in the spinal cords of rats across different groups to investigate the regulatory effects of EA on axon guidance signals. We found reduced levels of netrin-1, DCC and Rac1 in the spinal cord of rats in the SCI group. Netrin-1 is known to be highly expressed in the spinal neurons and oligodendrocytes of healthy adult rats (52), and the expression levels of DCC protein positively correlate with netrin-1 levels (53). Manitt et al. (54) reported that the levels of netrin-1 and DCC at the injury site in rats with SCI were significantly decreased, with this reduction persisting for up to 7 months post-injury. Furthermore, Nagendran et al. revealed (55) that the expression of netrin-1 was significantly downregulated after SCI, and that exogenous supplementation of netrin-1 could enhance DCC expression, thereby restoring the dendritic spine density of injured neurons to normal levels. Our findings align with these previous studies, as the expressions of netrin-1, DCC, and Rac1 in the spinal cord of rats in the EA group progressively increased with the duration of the intervention, and were significantly higher than those in the SCI group at each time point (day 7, 14, and 28). F-actin is a well-established downstream cytoskeletal effector of Rac1, mediating lamellipodium formation and growth cone dynamics. Our results showed that EA intervention significantly increased the fluorescence intensity of F-actin. Furthermore, a significant positive correlation was observed between Rac1 mRNA expression levels and F-actin fluorescence intensity. This suggests that EA may promote F-actin polymerization and cytoskeletal remodeling by upregulating Rac1 mRNA expression, thereby facilitating axonal repair. In summary, EA can promote the expression of nerve guidance signaling molecules and intracellular molecular switches in rats with SCI. The promoting effect of EA on the repair and regeneration of damaged nerves may be associated with the regulation of the netrin-1-mediated axon attraction signaling pathway.

In this study, EA treatment demonstrated significant and sustained beneficial effects. Motor function and fiber connections in the EA group showed marked improvement starting on the 7th day and persisting until the 28th day. Moreover, elevated levels of netrin-1, DCC, Rac1 and F-actin were observed after 28 days of EA intervention in the EA group compared to the SCI group. These discoveries suggest that EA treatment not only promotes nerve repair in the early stage following SCI, but also exerts a sustained intervention effect. Early intervention and long-term adherence to acupuncture treatment are crucial for promoting nerve repair after SCI. This study may provide references for the treatment cycle of EA for SCI. However, this study still has certain limitations. Firstly, a sham acupuncture control group was not included. Secondly, intervention periods beyond 28 days were not explored. Thirdly, the experimental findings were not validated through reverse approaches. Therefore, we suggest that future studies investigate whether longer treatment durations could further enhance functional recovery and include appropriate sham controls to confirm acupoint specificity. In addition, we will employ inhibitors or gene knockout models to explore the critical role of netrin-1 in promoting nerve repair in rats with SCI through EA.

Conclusions

In summary, EA at GV4 and GV14 can significantly improve the motor function of rats with SCI, regulate the nerve guidance factor netrin-1 and its receptor DCC, as well as the molecular switch Rac1, promote axonal cytoskeletal remodeling, and thereby achieve neural repair. The nerve repair effect of EA may be achieved by regulating the axon attraction signal pathway mediated by netrin-1. Initiating EA treatment for SCI as early as possible and extending the duration of the intervention are crucial for promoting nerve repair and restoring motor function in SCI rats.

Acknowledgments

We would like to thank BioRender for its support.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1816/rc

Data Sharing Statement: Available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1816/dss

Funding: This work was supported by the National Natural Science Foundation of China (Nos. 82374593 and 82305381); Shenzhen High-level Hospital Construction Fund (No. 24275G1001); the Fundamental Research Funds for the Central Universities (No. 2025-JYB-XJSJJ014); and the Independent Research Project of Postgraduates in Beijing University of Chinese Medicine (No. ZJKT2025016).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1816/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. BUCM-2024042505-2065) granted by the Animal Ethics Committee of Beijing University of Chinese Medicine, in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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