Healthy adult male SD rats (8 weeks of age, 200–220 g) were purchased from Shanghai Sipur-Bike Laboratory Animal Co., LTD (Shanghai, China, Animal license No.: SCXK(Shanghai) 2018-0006), and were raised in Laboratory Animal Center of Zhejiang Chinese Medicine University and approved by the China Laboratory Animal Management Evaluation and Accreditation Association (AAALAC, Animal license No.: SYXK(Zhejiang) 2018-0012). The rats were kept in controlled conditions and had free access to water and food. All animal experiments were conducted in accordance with all relevant animal testing and research ethics regulations and in accordance with the animal protocol approved by the Animal Ethics Committee of Zhejiang University of Chinese Medicine (ZSLL, 2017183).

A total of 144 rats were included in this study. All rats were randomly assigned to each group. (1) In the Sham group (n = 8), only the T10 vertebral plate was removed without any other intervention. (2,3,4) The SCI (n = 8), SCI-1d (n = 8) and SCI-7d (n = 8) groups were subjected to SCI model preparation without intervention. (5,6) SCI + phosphate buffer saline (PBS) (n = 8) and SCI + Chondroitinase ABC (ChABC) (Sigma-Aldrich, C3667, St. Louis, MO, USA) (n = 8) groups received 14 days of PBS and ChABC injections, respectively, based on the SCI model preparation. (7,8) After preparing the SCI model, the SCI + ShamEA (n = 8) (sham acupuncture control group, with needles hung off the skin without other interventions) and SCI + EA (n = 8) groups received acupuncture without electrical stimulation and acupuncture with electrical stimulation interventions for 14 days, starting on the second day. (9,10) SCI + Vehicle (Veh) (n = 8) and SCI + Chst11 (n = 8) groups were injected with empty vector control virus and Chst11 overexpression virus, respectively, and SCI models were prepared. (11) The SCI + Chst11 + EA (n = 8) group was added to the SCI + Chst11 group and included a 14-day EA intervention. The above section comprises a total of 88 rats, divided into 11 groups. Due to the unique nature of qRT-PCR experiments, this section is designated separately, involving a total of 56 rats. Groups included: Sham (n = 8), SCI (n = 8), SCI + PBS (n = 8), SCI + ChABC (n = 8), SCI + Veh (n = 8), SCI + Chst11 (n = 8), and SCI + Chst11 + EA (n = 8).

Dissolve ChABC or PBS in 1% fetal bovine serum (Sigma-Aldrich, F0193) to prepare a solution at a concentration of 5 U/mL [17]. Prepare fresh daily. Starting from day 1 after SCI model establishment, deliver ChABC or PBS to the rat’s spinal cord via intrathecal injection for 14 days [18].

Animal deaths and experimental dropouts inevitably occurred during this study, and this section provides an explanation. (1) Two rats in the Sham group died from postoperative infection complications. A rat sustained an accidental injury to its right hind limb during electromyography (EMG) amplitude measurement, which was not recorded. (2) One rat in the SCI group died from infection caused by bladder rupture during assisted urination due to excessive residual urine postoperatively. Additionally, one rat was lost during calcium imaging due to fiber optic detachment. (3) Two rats in the SCI + PBS group died from postoperative complications and bladder rupture. One rat was not recorded during calcium imaging due to fiber detachment. (4) Two rats in the SCI + ChABC group died from postoperative complications. Two rats were not recorded during calcium imaging due to fiber detachment. (5) In the SCI + ShamEA group, one rat failed to obtain amplitude measurements during EMG recording due to accidental injury to the right hindlimb muscles. (6) One rat in the SCI + EA group died from infection induced by postoperative bladder rupture. One rat failed to obtain amplitude measurements during EMG recording due to accidental injury to the right hindlimb muscles. (7) In the SCI + Veh, SCI + Chst11, and SCI + Chst11 + EA groups, two rats each (six total) died from bladder rupture during postoperative assisted urination. Among these, one rat in the SCI + Veh group could not be measured during qRT-PCR due to technical error. Due to technical errors, one rat each in the SCI + Chst11 and SCI + Chst11 + EA groups failed to yield data for the Chst11 gene loading lane, resulting in one dropout per group (total of 2). All animals removed from the groups were euthanized (Painless euthanasia was performed by administering 100–200 mg/kg of pentobarbital sodium solution via intraperitoneal injection to induce respiratory arrest in rats.) and promptly transported to the animal center for disposal by designated personnel.

Healthy adult male SD rats were deeply anesthetized with 3% pentobarbital sodium solution (Sigma-Aldrich, P3761) (2 mL/kg) and placed on a constant temperature operating table at 37 °C. The T10 cone was exposed, and the lamina was removed to expose the T10 spinal cord. An acute SCI model was created by impinging on the T10 spinal cord with a force of 200 kDynes using the IH Impactor 0400 SCI Impinger (Precision Systems and Instrumentation, LLC, IH-400, Lexington, KY, USA). The following signs indicated the successful modeling: (1) body spasmodic tremor, (2) spasmodic movement of the tail, and (3) intradural congestion or hematoma. The Basso–Beattie–Bresnahan (BBB) score was assessed on the day after modeling. Rats with scores $\ge$3 were excluded and supplemented under the same conditions. Postoperative treatment of rats that met the requirements of the acute SCI model included the following: (1) penicillin (North China Pharmaceutical Co., Ltd, 52123, Hebei, China) (100 U/d) was injected into the abdominal cavity for anti-infection for three consecutive days; (2) the room temperature was maintained at 20–25 °C; (3) the bladder was massaged twice a day until it appeared to urinate autonomously.

The EA was administered at the T9–T11 Jia-ji acupoints (EX-B2), located beside the spinous processes on the back, starting on the first day after surgery. The procedure was as follows: a disposable sterile stainless steel acupuncture needle (0.18 mm in diameter; Beijing Zhongyantaihe Medical Instrument Co., Ltd., V518215, Beijing, China) was inserted 1.5 mm lateral to the midline of the spinous process to a depth of 4–5 mm, until the needle tip contacted the lamina, and connected to the HANS200A electroacupuncture instrument (Nanjing Jisheng Medical Technology Co., Ltd., HANS200A, Nanjing, China). The parameters were set as follows: frequency, 2/100 Hz; current intensity, 1 mA; once daily for 20 min. The EA treatment period was 14 days.

As previously described [19], the BBB hind limb movement test was scored on a scale of 0–21 (0 = no visible hind limb movement; 21 = normal movement). Hind limb movement of rats with SCI was tested in an open field, including hind limb joint movement, weight support, footstep, coordination, paw position, fine paw movement, and trunk and tail control. BBB testing was performed on the first day after establishing the model. Each rat was placed in an open field and evaluated by two experimenters who were unaware of the experimental group for 3 min. One of the experimenters recorded the scores, and all rats were evaluated before establishing the SCI model to ensure baseline differences. Each score was averaged to obtain the final score. For specific scoring criteria, refer to Table 1.

The rats were bilaterally injected into the spinal cord with 500 nL of rAAV-CMV-Chst11-Flag-WPREs, AAV2/9 (BrainVTA Co., Ltd., PT-0634, Wuhan, China); a negative control rAAV-CMV-Flag-WPREs, AAV2/9 (BrainVTA Co., Ltd., PT-0634); or a combination of rAAV-CAG-FLEX-jGCaMP7f-WPRE-SV40pa, AAV2/9 (BrainVTA Co., Ltd., PT-1422) and rAAV-PV-CRE-bGHpa, AAV2/9 (BrainVTA Co., Ltd., PT-0275). The procedure was as follows: after exposing the spinal cord by removing the skin and connective tissue between the T9 and T10 vertebral plates with forceps, the virus was injected into the spinal cord under a microscope. Using the spinal vessels along the midline as anatomical landmarks, the injection site was positioned 1 mm lateral and 0.3 mm from the median vessels, with the needle inserted at a 10° angle. After injection, the needle tip was left in the spinal cord for 5 min to prevent viral backflow. The injection was performed at a final titer of 5.00 $\times$ 10¹² vg/mL, with a flow rate of 100 nL/min and a total volume of approximately 500 nL bilaterally, avoiding blood vessels and nerves.

The rats (n = 3 per group) were euthanized with 3% pentobarbital (Sigma-Aldrich, P3761) (100–200 mg/kg), followed by transmyocardial perfusion with saline, and the T10 segmental spinal cord was obtained, and immersed the spinal cord in a 4% paraformaldehyde solution (Sigma-Aldrich, 16005). The T10 spinal cord was cut into 12-µm-thick transverse sections using a Frozen Slicer (Thermo Scientific CryoStar™ NX70, 957010K, Thermo Fisher Scientific, Waltham, MA, USA), with each rat containing at least five consecutive sections. All slides were closed for 1 h at 37 °C in a water bath with TBST containing 10% normal goat serum, followed by overnight incubation at 4 °C with the primary antibody. Rabbit monoclonal anti-parvalbumin antibody (1:100, ab181086, Abcam, Cambridge, UK) and wisteria floribunda lectin (WFA) (1:100, B-1355-2, Vector Laboratories, Newark, California, USA) were used for incubation. The next day, the slices were rinsed with tris-buffered saline with tween-20 (TBST) and reacted with the corresponding secondary antibody mixture (1:200, Alexa Fluor® 555 Streptavidin Coupler and 1:300, Alexa Fluor® 488 Goat anti-rabbit IgG, Thermo Fisher Scientific). Fluorescence images were obtained using a Zeiss structured illuminated optical slice microscope (Axio Imager M2; Zeiss AG, Jena, Germany). All stained sections were observed and analyzed using a blinded method. Non-experimental personnel counted and statistically analyzed the number of cells in each section using Adobe Photoshop 13.0 (Adobe Systems, 13.0, San Jose, CA, USA).

Rats were euthanized with 3% pentobarbital (Sigma-Aldrich, P3761) (100–200 mg/kg), followed by transmyocardial perfusion with saline, and the T10 segmental spinal cord was obtained. On day 14 after SCI model preparation, rats were euthanized, and the T10 segmental spinal cord was rapidly removed. According to the manufacturer’s instructions, bicinchoninic acid assay (BCA; Pierce ™ BCA, 23227, Thermo Fisher Scientific) was used for protein concentration determination, with 20 mg protein per lane. To detect CS protein content, 5 U/mL of ChABC was added to the protein supernatant and incubated in a shaking bed at 37 °C for 8 h. Protein samples were separated on a 4%–15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Inc., IPVH00010, Billerica, MA, USA). The membrane was closed with 5% skim milk (Becton, Dickinson and Company, 232100, Franklin Lakes, NJ, USA) at room temperature for 1 h, and then incubated overnight at 4 °C in a diluted primary antibody in a closed buffer: $\beta$-actin (1:5000, Ms mAb to $\beta$-actin, Abcam), C4S (1:2000, mouse Anti-Chondroitin-4-Sulfate, MAB2030, Merck), C6S (1:2000, mouse Anti-Chondroitin-6-Sulfate, MAB2035, Merck), glutamic acid decarboxylase(GAD)65 and GAD67 (1:2000, Rb mAb to GAD65 + GAD67, ab183999, Abcam). The next day, the membrane was incubated with secondary antibodies (1:2000, goat anti-mouse, BK-M050, BIOKER, Hangzhou, China and 1:2000, goat anti-rabbit, 7074s, Cell Signaling Technology, Danvers, MA, USA) at room temperature for 2 h. Immunoreactivity was detected using enhanced chemiluminescence and visualized using an Image Quant LAS 4000 (Cytiva, LAS4000, Tokyo, Japan).

The rats were euthanized with 3% pentobarbital (100–200 mg/kg), followed by trans-myocardial perfusion with saline. Then, the T10 segmental spinal cord was obtained. A 0.5 cm segment of spinal cord tissue was placed in TRIzol reagent (Invitrogen, 15596018CN, Thermo Fisher Scientific) to extract total RNA. The extracted total RNA was reverse-transcribed using a transcription kit (Takara Biomedical Technology (Beijing) Co., Ltd., RR047A, Beijing, China) according to the manufacturer’s protocol. The target gene was mixed with SYBR Green (Solarbio, MQ10101S, Monad Biotech Co., Ltd., Beijing, China) reagent, and qRT-PCR was performed using LightCycler® 480 Instrument II (Roche, 05015243001, Basel, Switzerland). Gene expression was calculated using the 2^{-Δ⁢Δ⁢CT} method, and the final data were normalized to $\beta$-actin levels. The primer sequences are presented in Table 2.

Four weeks prior to modeling, rAAV-CAG-FLEX-jGCaMP7f-WPRE-SV40pa virus was bilaterally injected into the T9-T10 spinal cord junction using a Hamilton (Hamilton, 202630, Bonaduz, Switzerland) syringe. The injection flow rate was 100 nL/min, with a total injection volume of approximately 500 nL. The viral delivery sites and transfection coverage are shown in Supplementary Fig. 1. Immediately after successful model establishment, proceed with fiber implantation following these steps: The T10 spinal cord segment was fully exposed, and a 0.01 mm-thick perforated titanium plate (Xi’an Saite Metal Materials Development Co., Ltd., Xi’an, China) was placed on the spinal cord. The plate was secured to the surrounding soft tissue and vertebral body using non-absorbable surgical sutures at the four corners. All procedures were performed using a microscope. A fiber-optic probe (200 µm in diameter and 2 mm in length, Thinker Tech Nanjing Bioscience Inc, Nanjing, Jiangsu, China), mounted on a stereotaxic instrument (RWD, 68804, RWD Life Science, Shenzhen, China) with a fiber-optic holder, was positioned adjacent to the central canal of the spinal gray matter and inserted to a depth of 1.5 mm. The assembly was secured with dental cement (Shanghai New Century Dental Materials Co., Ltd., 20173170702, Shanghai, China) and adhesive (Single Bond Universal, 3 M ESPE, St. Paul, MN, USA) and covered with toner (Macklin, C805116, Shanghai, China) to prevent light interference. GCaMP7f fluorescence was excited at two wavelengths: 470 nm for the calcium-dependent signal and 405 nm for the baseline signal. The amplitude-modulated excitation light was reflected by a dichroic mirror and coupled to an optical fiber, enabling carrier wave fiber calcium imaging photometric recording. The recorded data were imported into MATLAB (MathWorks, R2018a, Natick, MA, USA) for analysis. Calcium activity was recorded from the first 2 s of the external stimulus to 10 s after the stimulus to capture changes in calcium signaling shortly after sensory induction (by pinching the foot).

Nicolet EDX: Viking QUEST equipment (Nicolet EDX: Viking QUEST, Natus Neurology, Madison, WI, USA) was used to acquire EMG data from the right lower limb gastrocnemius muscle of rats. The rats were placed on a special rat sleeve, and the hair of the gastrocnemius muscle was shaved using a hair-shaving machine. The rats were tested in an emotionally calm state. During detection, recording electrodes were inserted into the gastrocnemius muscle of the right lower limb of the rats, and EMG signals with a frequency range of 20–500 Hz were collected 20–25 times consecutively. The gastrocnemius potential was detected using the VikingSelect experimental system (Nicolet EDX: Viking QUEST, Viking QUEST v20.1.11, CA, USA), and the amplitude and duration were recorded to obtain average values.

All data were analyzed, and graphs were generated using GraphPad software (GraphPad, v8.0.2, San Diego, CA, USA) or MATLAB. The normality of the continuous variables was assessed using the Shapiro–Wilk test. For data conforming to a normal distribution, Student’s t-test (two-tailed) was used for comparisons between two groups, while one-way analysis of variance (ANOVA) was used for comparisons among multiple groups, followed by Tukey’s post hoc test. For data that did not follow a normal distribution, the Mann–Whitney U test and the Kruskal–Wallis H test were applied for comparisons between two groups and among multiple groups, respectively. A two-way ANOVA was performed to analyze BBB scores across groups. When an interaction between group (intervention) and time was observed, simple effects analysis was conducted and adjusted using Tukey’s post-hoc test. When no interaction between group (intervention) and time was observed, main effects analysis was performed followed by Tukey’s post-hoc test. All results are expressed as mean $\pm$ standard error of the mean (SEM). A p 0.05 was considered statistically significant.

We performed WB to detect CS, which is essential for the composition and function of the PNN, and its corresponding sulfated forms (C4S and C6S) (Fig. 1B,C). The results revealed that the expression levels of C4S and C6S in the spinal cord after SCI decreased on SCI-7d and increased on SCI-14d compared to the control group (Sham group) (Fig. 1B,C). The protein expression level of GAD can indirectly reflect the expression level of PV IN to a certain extent [20]. We examined the protein levels of both GAD isoforms after SCI (Fig. 1D). Both protein expressions were significantly reduced on SCI-7d, and GAD67 expression levels increased slightly on SCI-14d as the rat spinal cord repaired itself (Fig. 1E,F). Simultaneously, we analyzed the C4S/C6S ratio, and the results are presented in Fig. 1A. After SCI, this ratio began to decrease gradually, reaching its lowest point on SCI-7d. Subsequently, it began to increase slowly over time, becoming higher than that on SCI-1d by SCI-14d, although it remained lower than that in the control group.

Fig. 1.

The protein expression of CS and GAD was reduced following SCI. (A) Changes in the C4S/C6S ratio in normal rats at SCI-1d, SCI-7d, and SCI-14d (n = 4, F = 5.225, p = 0.0174). (B) Confirmation of two-dimensional gel electrophoresis results by WB. Analysis of C4S protein levels in normal rats at SCI-1d, SCI-7d, and SCI-14d, using $\beta$-actin as an internal control (n = 5, F = 34.73, p 0.0001). (C) Confirmation of two-dimensional gel electrophoresis results by WB. Analysis of C6S protein levels in normal rats at SCI-1d, SCI-7d, and SCI-14d, using $\beta$-actin as an internal control (n = 5, F = 34.37, p 0.0001). (D) Two-dimensional gel electrophoresis results for GAD67 and GAD65 in WB. (E) Confirmation of two-dimensional gel electrophoresis results by WB. Analysis of GAD67 protein levels in normal rats at SCI-1d, SCI-7d, and SCI-14d, using $\beta$-actin as an internal control (n = 5, F = 31.68, p 0.0001). (F) Confirmation of two-dimensional gel electrophoresis results by WB. Analysis of GAD65 protein levels in normal rats at SCI-1d, SCI-7d, and SCI-14d, using $\beta$-actin as an internal control (n = 5, F = 21.98, p 0.0001). *p 0.05. Data are expressed as mean $\pm$ SEM. Statistical analysis of the data results was performed using one-way ANOVA, followed by Tukey’s post hoc test. CS, chondroitin sulfate; GAD, glutamic acid decarboxylase; C4S, chondroitin 4-sulfation; C6S, chondroitin 6-sulfation; SCI, spinal cord injury; WB, Western blotting; ANOVA, analysis of variance.

To investigate the changes in PNN during SCI repair, we administered ChABC to the T10 region for 14 days. The results of the BBB scores revealed a significant interaction between group (intervention) and time (F_(9,84) = 192.6, p 0.0001). Analysis of main effects showed that group (intervention) significantly influenced BBB scores (F_(3,84) = 1544, p 0.0001). On day 14, the SCI + ChABC group scored significantly higher than the SCI + PBS group (p 0.0001). The main effects analysis also revealed a significant effect of time on BBB scores (F_(3,84) = 1706, p 0.0001). The SCI + ChABC group exhibited higher BBB scores on day 14 than on day 7 (p 0.0001). The BBB scores revealed that the motor function of the rats significantly improved after ChABC intervention (Fig. 2A). EMG results indicated that ChABC improved the SCI status, along with significant improvement in both EMG and muscle condition of the hind limbs of the rats (Fig. 2B–F). Meanwhile, WB analysis revealed that following ChABC administration, C4S and C6S protein expression was significantly elevated in the spinal cords of SCI + ChABC group rats, approaching CS expression levels observed in the normal rat spinal cord (Fig. 2G–I). After ChABC intervention, the C4S/C6S ratio was significantly higher in the SCI + ChABC group than in the remaining three groups on day 14, and a significant difference was observed between SCI + ChABC and SCI + PBS control groups (Fig. 2G), indicating improved PNN stability. The qRT-PCR results exhibited no significant alterations in the gene expression of the synthases synthesizing the different sulfated forms of CS (Fig. 2J,K), suggesting that ChABC intervention did not affect the gene expression profile of the relevant synthases.

Fig. 2.

ChABC increases CS protein expression after SCI and promotes motor function recovery. (A) Changes in BBB scores at 14 days post-establishment of the acute spinal cord injury model in the Sham group, SCI group, SCI + PBS group, and SCI + ChABC group (n = 6–7). There was an interaction between group (intervention) and time in the BBB scores. A two-way ANOVA was performed, followed by Tukey’s post hoc test. (B) Schematic representation of EMG waveforms recorded from different groups. (C) Statistical analysis of EMG amplitude in the gastrocnemius muscle of the Sham and SCI group (n = 5–6, t = 4.136, p = 0.0020). (D) Statistical graph of EMG duration in the gastrocnemius muscle of the hind limb in the Sham and SCI group (n = 6, t = 1.025, p = 0.3272). (E) Statistical graph of EMG amplitude in the gastrocnemius muscle of the hind limb in the SCI + PBS and SCI + ChABC group (n = 6, t = 2.596, p = 0.0267). (F) Statistical graph of EMG duration in the gastrocnemius muscle of the hind limb in the SCI + PBS and SCI + ChABC group (n = 6, t = 3.213, p = 0.0093). (G) Confirmation of two-dimensional gel electrophoresis results by WB. Changes in GAD67 and GAD65 protein expression and the C4S/C6S ratio in Sham, SCI, SCI + PBS, and SCI + ChABC groups (n = 5, F = 5.321, p = 0.0146). (H) Analysis of C4S protein levels in Sham, SCI, SCI + PBS, and SCI + ChABC groups, using $\beta$-actin as an internal control (n = 5, F = 19.64, p 0.0001). (I) Analysis of C6S protein levels in Sham, SCI, SCI + PBS, and SCI + ChABC groups using $\beta$-actin as an internal control (n = 5, F = 16.06, p 0.0001). (J) Synthetase Chst11 mRNA expression (n = 7–8, F = 8.911, p = 0.0003). (K) Synthetase Chst3 mRNA expression (n = 7–8, F = 6.806, p = 0.0014). ns: not significant, ns: not significant, *p 0.05. Data are expressed as mean $\pm$ SEM. (C), (D), (E), and (F) were analyzed using Student’s t-test (two-tailed) to compare the two groups. (G), (H), (I), (J) and (K) were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. ChABC, Chondroitinase ABC; PNN, perineuronal net; BBB, Basso–Beattie–Bresnahan; EMG, electromyography; Chst11, carbohydrate sulfotransferase-11; Chst3, carbohydrate sulfotransferase-3. Fig. 2B was created using Adobe Illustrator 2019 ( 23.0.3 x64, Adobe Systems Incorporated, San Jose, CA, USA).

Changes in CS are closely related to the progression of SCI repair, and the ratio of C4S to C6S affects PNN structure stability after SCI. Changes in CS affect the activity and expression of PV IN. We injected AAV indicative of changes in calcium signaling and labeled PV IN in the spinal cord of rats at the junction of T9 and T10. We detected and recorded calcium signals on day 14 after preparing the SCI model (Fig. 3A,B). Our previous study confirmed that neurons labeled by the jGCamp7f virus are PV IN, and the virus co-infects with PV [21]. We also validated the virus in rats and demonstrated that jGCaMP7f binds to PV IN with a significant effect (Supplementary Fig. 1). After ensuring that the surroundings were quiet and suitable and that the rats were emotionally stable, we recorded calcium signals from 2 s before stimulation to 10 s after stimulation. The results (Fig. 3C,D) revealed that the amplitude of calcium signaling in PV IN was significantly reduced in the SCI group than in the control group (Fig. 3E), and the peak calcium signal during neuronal activity decreased (Fig. 3F). The amplitude of calcium signaling in PV IN at the injury site was significantly increased in SCI + ChABC than in SCI + PBS (Fig. 3E), with no significant increase in the peak of calcium signal (Fig. 3F). WB results (Fig. 3G) revealed that the GAD67 expression level was significantly increased in the SCI + ChABC group than in the SCI + PBS control group, with no significant difference in GAD65 expression among the groups (Fig. 3H,I).

Fig. 3.

ChABC enhances PV IN activity and GAD protein expression after SCI. (A) Schematic diagram of the fiber-optic placement in the rat spinal cord. (B) Schematic timeline of AAV virus injection and fiber-optic placement. (C) Heatmaps illustrating calcium signal changes in Sham, SCI, SCI + PBS, and SCI + ChABC groups recorded after excitation at 470 nm calcium-related signal wavelength. (D) Schematic diagram of folded calcium signal changes in Sham, SCI, SCI + PBS, and SCI + ChABC groups, reflecting changes in PV IN activity. (E) Statistical graph of the mean calcium signal change (n = 4–6, F = 30.53, p 0.0001). (F) Comparison of calcium signal peak frequencies among the groups (n = 4–6, F = 26.21, p 0.0001). (G) Changes in GAD67 and GAD65 protein expression in Sham, SCI, SCI + PBS, and SCI + ChABC groups. (H) Statistical graph of GAD67 expression (n = 5, F = 30.69, p 0.0001). (I) GAD65 protein expression statistics (n = 5, F = 2.078, p = 0.1435). ns: not significant, *p 0.05. Data are expressed as mean $\pm$ SEM. Statistical analysis of the data results was performed using one-way ANOVA, followed by Tukey’s post hoc test. PV, parvalbumin; AAV, adeno-associated virus; PV IN, parvalbumin interneuron. Fig. 3A was created using Adobe Illustrator 2019 ( 23.0.3 x64, Adobe Systems Incorporated)

Previous studies have demonstrated that ChABC significantly promotes PNN stability and enhances PV IN activity. Subsequently, we used EA to intervene in SCI rats and verified its therapeutic effects to determine whether the repair effect of EA on SCI was similar to or significantly different from that of ChABC. The results of BBB scoring revealed a significant interaction between group (intervention) and time (F_(9,96) = 167.9, p 0.0001). Analysis of main effects indicated that group (intervention) significantly influenced BBB scores (F_(3,96) = 1323, p 0.0001). On day 14, the SCI + EA group scored significantly higher than the SCI + ShamEA group (p = 0.0011). The main effects analysis also revealed a significant effect of time on BBB scores (F_(3,96) = 1578, p 0.0001). The SCI + EA group exhibited higher BBB scores on day 14 than on day 7 (p = 0.0073). Motor function was significantly improved in rats after EA intervention (Fig. 4A). EMG signal detection results indicated that the amplitude and duration were significantly higher in the SCI + EA group than in the SCI + ShamEA group (Fig. 4B,F,G). EA significantly promoted SCI repair, effectively increased the expression levels of C4S and C6S (Fig. 4C–E), and improved PNN stability (Fig. 4C). The GAD protein expression results were consistent with those of CS (Fig. 4H–J). EA significantly increased CS and GAD expression and promoted SCI repair.

Fig. 4.

EA increased the protein expression levels of CS and GAD after SCI and improved the relative stability of the PNN structure. (A) BBB motor function scores of Sham, SCI, SCI + ShamEA, and SCI + EA groups at 7 and 14 days after acute SCI model preparation (n = 7). There was an interaction between group (intervention) and time in the BBB scores. A two-way ANOVA was performed, followed by Tukey’s post hoc test. (B) Schematic diagram of the waveforms of EMG signals measured in SCI + ShamEA and SCI + EA groups. (C) Protein expression of C4S and C6S and variation of C4S/C6S in Sham, SCI, SCI + ShamEA, and SCI + EA groups (n = 4, F = 9.189, p = 0.0020). (D,E) Confirmation of two-dimensional gel electrophoresis results by WB. Analysis of C4S and C6S protein levels in Sham, SCI, SCI + ShamEA, and SCI + EA groups, using $\beta$-actin as an internal control (C4S: n = 5, F = 41.97, p 0.0001. C6S: n = 5, F = 53.71, p 0.0001). (F,G) Statistical plots of amplitude versus duration after electromyographic testing of hindlimb gastrocnemius muscle in SCI + ShamEA and SCI + EA groups (Amplitude: n = 6–7, t = 3.087, p = 0.0103. Duration: n = 7–8, t = 2.400, p = 0.0321). (H) WB plots of GAD67 and GAD65 in Sham, SCI, SCI + ShamEA, and SCI + EA groups. (I,J) Confirmation of two-dimensional gel electrophoresis results by WB. Analysis of GAD67 and GAD65 protein levels in Sham, SCI, SCI + ShamEA, and SCI + EA groups, using $\beta$-actin as an internal control (n = 5, GAD67: F = 27.07, ns: not significant, p 0.0001. GAD65: F = 24.81, p 0.0001). *p 0.05. Data are expressed as mean $\pm$ SEM. (F) and (G) were analyzed using Student’s t-test (two-tailed) to compare the two groups. (C), (D), (E), (I) and (J) were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. EA, electroacupuncture. Fig. 4B was created using Adobe Illustrator 2019 ( 23.0.3 x64, Adobe Systems Incorporated)

When normal rats contained more Chst11 than the normal range, their PNN inhibitory properties were enhanced, and the rate of spinal cord repair was inhibited following SCI. Based on this characterization, we injected the virus overexpressing Chst11 into the spinal cord of rats at the junction of T9 and T10 28 days prior, and it was fully expressed in the T10 segment (Fig. 5A,B). Subsequently, we verified the efficiency of Chst11 overexpression (Fig. 6C,D). To investigate the potential of EA in promoting SCI repair by inhibiting Chst11 and modulating CS and GAD expression, we administered EA with a virus containing Chst11 to rats for 14 days, followed by detection and analysis of T10 spinal cord segments. The results of BBB scores revealed a significant interaction between group (intervention) and time (F_(6,60) = 6.563, p 0.0001). Analysis of main effects showed that group (intervention) significantly influenced BBB scores (F_(2,60) = 18.53, p 0.0001). At Day 14, the SCI + Chst11 + EA group scored significantly higher than the SCI + Chst11 group (p 0.0001). The main effects analysis also revealed a significant effect of time on BBB scores (F_(3,60) = 1264, p 0.0001). The SCI + Chst11 + EA group exhibited higher BBB scores on day 14 than on day 7 (p = 0.0011). EA reversed the inhibitory effect of Chst11, and motor function was significantly improved in the SCI + Chst11 + EA group (Fig. 5C). The amplitude of the hindlimb unit EMG was significantly higher in the SCI + Chst11 + EA group than in the SCI + Chst11 group (Fig. 5K–M). C4S and C6S protein expression levels were significantly higher in the SCI + Chst11 + EA group than in the SCI + Chst11 group, and the inhibitory properties of Chst11 were reversed by EA, promoting SCI recovery (Fig. 5D–F). The EA reversed Chst11 inhibition and improved PNN stability (Fig. 5G). GAD67 and 65 also displayed similarities to C4S and C6S protein expression (Fig. 5H–J).

Fig. 5.

EA reversed the inhibitory effect of Chst11 and increased the protein expression levels of CS and GAD. (A) Schematic diagram illustrating AAV injection. (B) Timeline of AAV virus injection and SCI model preparation. (C) Statistical graphs of BBB scores of SCI + Veh, SCI + Chst11, and SCI + Chst11 + EA groups at 7 and 14 days after SCI model preparation (n = 6). There was an interaction between group (intervention) and time in the BBB scores. A two-way ANOVA was performed, followed by Tukey’s post hoc test. (D) C4S and C6S protein expression in SCI + Veh, SCI + Chst11, and SCI + Chst11 + EA groups at day 14 after SCI (n = 5). (E,F) Statistical graphs of C4S and C6S protein expression (n = 5. C4S: F = 7.805, p = 0.0067. C6S: F = 10.23, p = 0.0026). (G) Changes in the C4S/C6S ratio in SCI + Veh, SCI + Chst11, and SCI + Chst11 + EA groups (n = 4, F = 4.880, p = 0.0367). (H) GAD67 and GAD65 protein expression in SCI + Veh, SCI + Chst11, and SCI + Chst11 + EA groups on day 14 after SCI (n = 5). (I,J) Statistical graphs of GAD67 and GAD65 protein expression (n = 5. GAD67: F = 21.16, p = 0.0001. GAD65: F = 20.70, p = 0.0001). (K) Schematic diagrams of waveforms obtained by EMG in SCI + Veh, SCI + Chst11, and SCI + Chst11 + EA groups. (L,M) Statistical graphs of EMG signals of the gastrocnemius muscle in the hind limbs of rats in SCI + Veh, SCI + Chst11, and SCI + Chst11 + EA groups (n = 6. Amplitude: F = 36.06, ns: not significant, p 0.0001. Duration: F = 0.8760, p = 0.4367). (N,O) mRNA expression of Chst11 and Chst3 in the three groups on day 14 after SCI (Chst11: n = 5, F = 3.030, p = 0.0860. Chst3: n = 5–6, F = 1.192, p = 0.3327). *p 0.05. Data are expressed as mean $\pm$ SEM. (E), (F), (G), (I), (J) and (L–O) were performed using one-way ANOVA, followed by Tukey’s post hoc test. Fig. 5A,K were created using Adobe Illustrator 2019 ( 23.0.3 x64, Adobe Systems Incorporated)

Fig. 6.

EA reversed the Chst11-induced reduction in the number of PNN and PV IN positive cells. (A) Representative fluorescence plots of WFA + PV+ in SCI + Veh, SCI + Chst11, and SCI + Chst11 + EA groups at day 14. (Scale bar = 100 µm and 10 µm). White arrows indicate relevant positive staining points. The white box marks the rightmost magnified area. (B) Statistical graph of the number of WFA + PV + positive cells in SCI + Veh, SCI + Chst11, and SCI + Chst11 + EA groups illustrated by IF. Each rat contains 5 sections. Statistical analysis of the data results was performed using one-way ANOVA, followed by Tukey’s post hoc test (n = 3, F = 141.3, p 0.0001). (C) Chst11 protein expression in SCI + Veh and SCI + Chst11 groups on day 14 after SCI. (D) Statistical graph of Chst11 protein expression in SCI + Veh and SCI + Chst11 groups. Statistical analysis of the data results was performed using Student’s t-test (two-tailed) (n = 6, p = 0.0297). *p 0.05. Data are expressed as mean $\pm$ SEM. WFA, wisteria floribunda agglutinin; IF, immunofluorescence.

The results of qRT-PCR similarly indicated that EA did not alter the endogenous expression of CS-related synthase genes in rats while promoting SCI recovery, suggesting that its impact on the organism was minimal (Fig. 5N,O). IF results were consistent with those of WB, and EA effectively increased the number of PNN and PV IN after SCI, facilitating SCI repair (Fig. 6A,B). The original western blot data for this study are provided in the Supplementary Materials-WB.