All animal experiments were performed in compliance with all relevant ethical regulations for animal testing and research and in accordance with animal protocols approved by the Ethics Committee of Shanghai General Hospital (No. 2023AW004). Healthy adult male SPF-grade Sprague–Dawley (SD) rats (8–10 weeks old, 250–300 g body weight) were obtained from and housed in the Experimental Animal Centre of Shanghai General Hospital. Rats were maintained under controlled conditions (24 ± 3 °C, 12-h day/night cycle) with access to food and water ad libitum. We ensured that all rats received proper care during and after the surgery. The rats were randomly divided into three groups: the Sham group (n = 15) (undergoing laminectomy only and executed at 28 days), the SCI group, and the SCI + EA group. The SCI and the SCI + EA groups were further divided into five subgroups based on the duration of 1 day (n = 30), 3 days (n = 30), 7 days (n = 30), 14 days (n = 30), and 28 days (n = 30). Among them, 55 rats were allocated for qPCR and RNA-Seq, 55 for western blot analysis, and the remaining 55 for immunofluorescence and immunohistochemistry experiments. All the experimental protocols strictly followed the guidelines of the National Institutes of Health (NIH) on the use of laboratory animals.

A complete transverse incision was performed on the right side of the rats’ spinal cord (Cheriyan et al. 2014). Zoletil 50 was diluted 1:10 in saline and administered via intraperitoneal injection at a dose of 1 mL/100 g of body weight to induce anesthesia. A median longitudinal incision of about 2 cm was made in the back of the rat, centered on the T10 spinous process to expose and remove the T10 vertebral plate. This exposed about 5 mm of spinal cord segments, and the right spinal cord was cut by the midline of the spinal cord with microscopic scissors. Then, the rats were returned to clean and warm home cages until they fully recovered and were given adequate food and water. All animals were injected with Gentamicin sulfate 20 mg intraperitoneally for 3 days. Manual bladder expression was performed every 8–12 h to assist bladder emptying until voluntary urinary function was restored. Rats with BBB behavioral scores of 0–2 were included in the experiments.

Rats were subjected to EA treatment at the T9–T11 Jiaji (EX-B2) acupoints, which were located on the two sides of the spinous process of the dorsal part. All sterilized disposable stainless steel acupuncture needles (0.25 mm * 25 mm, Suzhou Tianxie Acupuncture Instruments Co. Ltd.) were inserted as deep as 5–8 mm. Subsequently, an EA apparatus (Changzhou Yingdi Acupuncture Instruments Co. Ltd.) was connected with parameters set as follows: a frequency of 2 Hz and the current intensity adjusted to induce slight muscle contraction around the treatment area, lasting for 20 min. The initial treatment was given to rats after they woke up and achieved a BBB score of 0–2. For SCI + EA groups, treatment was given once daily. Rats in the SCI + EA groups receiving EA treatment were positioned on a platform measuring 5 cm * 8 cm * 50 cm, while SCI groups of rats were placed on the platform for 20 min without EA intervention.

The Basso–Beattie–Bresnahan test (BBB) is judged on a scale of 0–21 points (0, complete hind-limb paralysis; 21, normal locomotion) and allows for a comprehensive evaluation of overall locomotor performance and hind-limb recovery through behaviors such as weight support, joint mobility, and tail control (Basso et al. 1995). Briefly, each rat was placed in an open field and evaluated by two experimenters who were blinded to experimental groups and counted the scores. The rats' footprints were analyzed by dipping their front paws in a green, non-toxic dye and their hind paws in a red non-toxic dye (Brown et al. 1989). The rat was trained before the gait analysis experiment to pass through a narrow tunnel (100 cm * 7 cm * 7 cm) and reach a dark box containing its cohabiting partner and food. If the rat failed to pass through the tunnel within 3 s or stopped inside the tunnel, the test was repeated. After completing the treatment cycle, all rats underwent gait testing. Footprints within the middle 70 cm of the journey were chosen for analysis to ensure the rats were moving at a consistent speed. The angle of rotation of the hindfoot and the coefficient of motor coordination of the fore and hind limbs were measured using the methodology from a previous study (Stirling et al. 2004).

After each treatment period, the rats were anesthetized with Zoletil 50 and then transcardially perfused with 100–150 mL of saline through the left ventricle, following the opening of the right atrium. Spinal cord tissues were then dissected, and segments 0.5 cm above and below the injury site were collected and stored at − 80 °C for later use. There were 10 rats in the Sham group and 20 rats each in the 1-day, 3-day, 7-day, 14-day, and 28-day groups for RNA sequencing, qPCR, and western blotting. Additionally, 55 rats were selected for the collection of frozen tissue sections. After saline perfusion, the SCI segment was perfused with 250 mL of 4% paraformaldehyde (Servicebio, Wuhan, China) and left in 4% paraformaldehyde overnight.

Tissues were collected from the SCI region, and total RNA was extracted using the Trizol reagent (Invitrogen, CA, USA). The cDNA was synthesized and analyzed using the kit (Vazyme, Nanjing, China). RT-qPCR was performed using SYBR Green Master Mix (Vazyme, Nanjing, China) and processed in a Bioer device. Primers for qPCR were obtained from Sangon Biotech (Shanghai, China) (Table 1).

Spinal cord tissues of rats were homogenized with lysis buffer containing protease inhibitor and phosphatase inhibitor (Epizyme, Shanghai, China). After sonication and centrifugation, the supernatant was collected, and the total protein concentration of each sample was determined using the bicinchoninic acid (BCA) method according to the kit’s instructions. Furthermore, equal amounts of protein from each sample were loaded on 10% SDS-PAGE gels and transferred to PVDF membranes (Epizyme, Shanghai, China). After the transfer, the membrane was incubated in blocking solution (5% skimmed milk) and incubated overnight at 4 °C in appropriately diluted primary antibody solution: Anti-UCN2 (1:1000, ab231050, Abcam), Anti-Phospho-PKA C (1:1000, 4781S, Cell Signaling Technology), Anti -PKA C (1:1000, 4782S, Cell Signaling Technology), Anti-Phospho-NF-κB (1:1000, 3033S, Cell Signaling Technology), Anti-NF-κB (1:1000, 8242T, Cell Signaling Technology), Anti-NMDAR1 (1:1000, ab109182, Abcam), Anti-Phospho-CREB (S133) (1:750, GB114684-100, Servicebio), Anti-CREB (1:750, GB11539-100, Servicebio), and Anti-Actin (1:1000, GB113225-100, Servicebio). The next day, the sample was washed in TBST, probed with specific secondary antibodies: Anti-rabbit IgG (1:2000, 7074S, Cell Signaling Technology), and detected using the ECL detection system (Epizyme, Shanghai, China). Densitometry analysis of bands was done using iBright FL1000 imaging software (Thermo Fisher Scientific, USA).

Transverse spinal cord specimens fixed in 4% paraformaldehyde were successively submerged in 15% and 30% sucrose solutions to undergo dehydration. Following this, the specimens were methodically sliced into longitudinal sections (8-µm thick) using a cryostat (Thermo, NX50, USA) and perfused with PBS. Sections were blocked in blocking buffer (Beyotime, Shanghai, China) and incubated overnight at 4 °C with primary antibodies: Anti-β-Tubulin III antibody (1:100, T2200, Sigma-Aldrich), Anti-GFAP (1:100, MAB360, Millipore), Anti-Myelin Basic Protein Antibody (1:100, MAB386, Sigma-Aldrich), Anti-Iba1 (1:100, 019-19741, Wako), and Anti-Neurofilament 200 antibody (1:100, N4142, Sigma-Aldrich). The following day, samples were incubated for 2 h with secondary antibodies: Goat anti-Mouse IgG 546 (1:500, A-11030, Invitrogen), Goat anti-Rabbit IgG 488 (1:500, A32731, Invitrogen), and Goat anti-Rat IgG (1:400, A0507, Beyotime). Cell nuclei were stained with DAPI. Images were visualized using a fluorescence microscope (Olympus, Japan) and analyzed using ImageJ software.

The frozen sections were rinsed three times with PBS, each for 5 min, followed by an antigen fixation and blocking in 3% BSA solution. All sections were incubated overnight at 4 °C with primary antibody: Anti-UCN2 (1:100, ab231050, Abcam). The following day, samples were incubated for 50 min with secondary antibody: Goat Anti-rabbit (1:200, G1302, Servicebio), and freshly concocted DAB chromogenic solution was applied to the sections. After rinsing the sections to stop color development, counterstaining with hematoxylin was performed for about 3 min. This was followed by dehydration, mounting, and observation under a microscope. Images were analyzed by ImageJ software.

The bar graphs were generated using GraphPad Prism 9.0 software, and the analysis of the area expressing positive results for immunofluorescence and immunohistochemistry was conducted using Image J software. The statistical significance of the difference between the control and experimental groups was determined by one-way ANOVA or two-way ANOVA followed by Tukey’s post hoc tests, which were performed with SPSS 19.0 (SPSS, Inc., Chicago, USA). Data are shown as mean ± SEM and considered to indicate statistically significant if p < 0.05.