Adult C57BL/6 mice (18–20 g, 8–10 weeks old) were purchased from Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (Taizhou, Jiangsu, China; License No.: SCXK (China, Jiangsu) 2020-0009) and housed in the Key Laboratory of Integrated Traditional Chinese and Western Medicine, Anhui University of Chinese Medicine. The animal experiments were approved by the Ethics Committee of our institute (Approval No.: AHUCM-mouse-2024224). Mice were kept in cages (five per cage) under a 12-hour light/dark cycle, controlled temperature (23–25 °C), and 40–50% humidity, with free access to food and water.

In total, 230 mice were randomly assigned to groups using a randomized digital table. Data collection and analysis were performed by blinded researchers. The experimental groups were as follows: (i) Control group (Control), (ii) SCI-vehicle group (SCI-Veh), (iii) SCI-LTG group (SCI-LTG), (iv) SCI-Bafilomycin A1 (Baf) group (SCI-Baf), and (v) SCI-LTG-Baf group (SCI-LTG-Baf). The Control group underwent laminectomy without spinal cord transection as a sham procedure, while all other groups underwent spinal cord transection. The SCI-LTG group received LTG treatment, the SCI-Veh group received an equivalent volume of DMSO and saline as the vehicle, and the SCI-Baf group was treated with Bafilomycin. The SCI-LTG-Baf group received both LTG and Bafilomycin. The number of mice in each experiment is provided in the figure legend.

LTG (3,5-diamino-6-[2,3-dichlorophenyl]-1,2,4-triazine, L3791, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO (Dimethyl sulfoxide, 276855, Sigma-Aldrich) and diluted to 30 mg/mL with 0.9% saline. Mice were administered LTG via intraperitoneal injection at a dose of 30 mg/kg immediately following SCI surgery, with daily injections continuing for 4 weeks [18, 20]. Bafilomycin A1 (Baf, 11038, Cayman Chemical, Ann Arbor, MI, USA) was dissolved in DMSO and adjusted to 0.5 mg/mL with 0.9% saline. Baf was injected intraperitoneally at a dose of 1 mg/kg [21], beginning 1 h after the LTG injection and continuing daily for the same 4-week period. The control group received a vehicle solution with the same dilution components to assess any potential effects of the solvent.

Mice (8–10 weeks old, 20–25 g) underwent surgery while under 40 mg/kg pentobarbital sodium (P3761, Sigma-Aldrich) anesthesia (i.p.). For the transection groups, a complete spinal cord transection at T9 was performed using iridectomy scissors and a micro-knife to ensure thorough lesioning. The muscle, fascia, and skin were then sutured [10, 22]. Post-surgery, manual urination was carried out twice daily until bladder function was restored. All procedures were performed by a blinded, independent surgeon.

Following pentobarbital sodium anesthesia (40 mg/kg, i.p.), mice received a saline perfusion, followed by 0.1 M phosphate-buffered saline (PBS, P5493, Sigma-Aldrich) containing 4% paraformaldehyde (PFA, pH 7.4, 441244, Sigma-Aldrich). The spinal cords were post-fixed in 4% PFA overnight at 4 °C, transferred to 20% sucrose (S5016, Sigma-Aldrich) for 12 h, followed by 30% sucrose for another 12 h, and finally embedded in optimum cutting temperature (OCT) compound (4583, Sakura Finetek USA, Torrance, CA, USA). Tissues were sectioned at 25 µm and incubated with primary antibodies overnight at 4 °C (NeuN, a neuronal nuclear-specific protein, ABN78, Millipore, Burlington, MA, USA, 1:500; GFAP-Cy3, glial fibrillary acidic protein-cyanine 3, ab49874, Abcam, Cambridge, UK, 1:1000; ChAT, choline acetyltransferase, AB144, Sigma-Aldrich, 1:200; vGlut1, vesicular glutamate transporter 1, AB5905, Sigma-Aldrich, 1:500; p62, sequestosome-1, P0067, Sigma-Aldrich, 1:500; LC3B, B-light chain 3, NB100-2220, Novus, Centennial, CO, USA, 1:500). The sections were then washed and incubated overnight at 4 °C with Alexa Fluor 488 or 555-conjugated secondary antibodies (A31572, A21206, Thermo Fisher Scientific, Waltham, MA, USA, 1:600), followed by observation with a Zeiss LSM710 confocal microscope (Zeiss, Oberkochen, Germany). Quantification was performed using ImageJ software (1.43, NIH, Bethesda, MD, USA), with researchers blinded to the group allocations during analysis.

To prepare samples for Western blot analysis, 1.5 mm of spinal cord tissue containing the lesion scar was collected from each mouse after euthanizing with 200 mg/kg pentobarbital sodium (i.p.) and perfusion with ice-cold PBS to clear blood. Samples were sonicated and lysed in radioimmunoprecipitation assay (RIPA) buffer (1% nonidet P-40, NP-40, ST2045, Beyotime, Shanghai, China; 50 mM tris hydroxymethyl-aminomethane hydrochloride (Tris-HCl, B548127, Sangon Biotech, Shanghai, China); 1 mM disodium ethylenediaminetetraacetate (Na₂-EDTA, E8030, Solarbio Life Sciences, Beijing, China); 0.25% sodium deoxycholate (Na-deoxycholate, ST2049, Beyotime); 150 mM sodium chloride (NaCl, A501218, Sangon Biotech)) containing protease and phosphatase inhibitors (4906837001, Roche, Basel, Switzerland). After pretreatment, equal protein amounts were separated by SDS-PAGE and transferred to 0.2 µm polyvinylidene fluoride (PVDF) membranes (ISEQ00010, Millipore), followed by overnight incubation at 4 °C with primary antibodies (p62, P0067, Sigma-Aldrich, 1:1000; LC3B, NB100-2220, Novus, 1:1000; vGlut1, 48-2400, Invitrogen, Carlsbad, CA, USA, 1:500; Bcl-2, B-cell lymphoma-2, ab182858, Abcam, 1:1000; Bax, Bcl-2-associated x, ab32503, Abcam, 1:1000; Cleaved caspase-3, ab32351, Abcam, 1:1000; $\beta$-actin, A1978, Sigma-Aldrich, 1:1000; GAPDH, G9545, Sigma-Aldrich, 1:1000). The membranes were treated with HRP-conjugated secondary antibodies (#7074, Cell Signaling Technology, Danvers, MA, USA, 1:5000) and subsequently exposed to a chemiluminescence reagent (WBKLS0500, Millipore) for protein detection. Signal intensity was analyzed using ImageJ software (1.43, NIH), with researchers blinded to group allocation during analysis.

To observe apoptotic neurons, a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) assay was performed using the One Step TUNEL Apoptosis Assay Kit (C1086, Beyotime) according to the manufacturer’s instructions. After tissue preparation and freezing, the sections were post-fixed with 4% PFA for 30 minutes, then treated with PBS containing 0.5% Triton X-100 (X100, Sigma-Aldrich) and incubated at room temperature for 5 minutes. Sections were subsequently incubated with 50 µL TUNEL reaction mixture at 37 °C for 1 h in a dark environment. Finally, the images were captured, and TUNEL⁺ neurons were manually counted using ImageJ software (1.43, NIH) in a blinded manner.

Hindlimb movement recovery was assessed at 0, 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, and 63 days using the Basso Mouse Scale (BMS) open-field test, as described by Basso and colleagues [23]. One day before SCI surgery, mice were positioned in the open field to familiarize themselves with the environment. BMS scoring began on the first-day post-surgery, with each session lasting 4 minutes. The BMS scale ranges from 0 for complete paralysis, to 9 for normal hindlimb movement. A higher score indicates better recovery. Three mice were excluded from the experiment because their hindlimbs were bitten and injured after SCI. Two researchers, blinded to group allocation, independently evaluated the mice. When the BMS scores varied between hind limbs, the average was calculated.

To quantify the lesion site area, spinal cords were sectioned sagittally, with the midline as the reference point. Three sagittal sections centered around the spinal cord midline were selected for analysis. Each section was immunostained with GFAP-Cy3 to delineate the boundary of the lesion site, defined as the region lacking intact GFAP⁺ astrocytic networks. The lesion area for each selected section was manually outlined and measured with ImageJ software. The average lesion area across the three sections was calculated, representing the lesion site area for each sample [24].

All quantification was performed blind to group allocation, with sample numbers provided in the corresponding figure legends. Data are presented as the mean $\pm$ standard error of the mean (SEM). All data were analyzed with GraphPad Prism 10 (10.1.2, GraphPad Software, Columbia, MD, USA). Before applying parametric statistical tests, data normality was assessed using the Shapiro-Wilk test. If the data were normally distributed (p $>$ 0.05), parametric tests were applied; otherwise, non-parametric tests were used where appropriate. The results of the Shapiro-Wilk test showed that the data were normally distributed (p $>$ 0.05); thus, parametric tests were used. For statistical analysis, Student’s t-test was used for single comparisons between two groups. In addition, multiple comparisons were conducted using one-way ANOVA followed by Bonferroni correction or two-way ANOVA with Fisher’s least significant difference (LSD) post hoc test. Significance thresholds are indicated as *p 0.05, **p 0.01, ***p 0.001, or ****p 0.0001.

We conducted a series of experiments to evaluate the neuroprotective effect of LTG (Fig. 1a). Spinal cords were harvested and serially sectioned into coronal slices that spanned 600 µm rostrally and caudally around the lesion epicenter (Fig. 1b). The results showed that LTG treatment (30 mg/mL) significantly improved the survival of spinal neurons 4 weeks after SCI, suggesting it has the potential to mitigate neuronal loss (Fig. 1c,d). Further analyses revealed an increase in ChAT⁺ motor neurons in the SCI-LTG group compared with the SCI-Veh group at 4 weeks post-SCI, thus reinforcing the neuroprotective effect of LTG (Fig. 1e,f). Additionally, co-immunostaining for 4^′-6-diamidino-2-phenylindole (DAPI) and GFAP was performed to assess the lesion area at the injury site (Fig. 1g,h). This revealed a smaller lesion area in the SCI-LTG group compared with the SCI-Veh group. Overall, LTG treatment effectively reduced spinal neural injury after SCI.

Fig. 1.

LTG mitigates post-traumatic spinal neural injury. (a) Experimental timeline showing SCI surgery, LTG treatment, neuroprotection assessment, BMS scoring, analysis of apoptosis and autophagy-related proteins, and synapse survival post-injury. (b) A representative serial coronal section showing NeuN⁺ and ChAT⁺ neurons, and spanning rostrally (–600 µm) and caudally (+600 µm) from the lesion epicenter. (c,d) Co-immunostaining (c) and quantification (d) of NeuN and GFAP from 600 µm rostral to 600 µm caudal around the epicenter in SCI-Veh and SCI-LTG groups at 4 weeks post-injury. The number of NeuN⁺ neurons was counted, with data presented as mean $\pm$ SEM of 5 independent experiments, with one mouse per group in each independent experiment. (n = 5 per group). Two-way ANOVA, Fisher’s LSD. **p 0.01. The scale bar in the figure is 200 µm. (e,f) Immunolabeling (e) and quantification (f) of ChAT⁺ motor neurons spanning 600 µm rostrally and caudally from the lesion epicenter in SCI-Veh and SCI-LTG groups 4 weeks after SCI. The number of ChAT⁺ motor neurons was counted and data are presented as mean $\pm$ SEM of 5 independent experiments, each with one mouse per group. (n = 5 per group). Two-way ANOVA, Fisher’s LSD. *p 0.05, **p 0.01. The scale bar in the figure is 100 µm. (g,h) Co-immunostaining (g) and quantification (h) of GFAP and DAPI showing the lesion site area in SCI-Veh and SCI-LTG groups at 4 weeks post-injury. Lesion site area is presented as the mean $\pm$ SEM from 5 independent experiments, each with one mouse per group (n = 5 per group). Student’s t-test. **p 0.01. The scale bar in each panel is 100 µm (left panels), 50 µm (right panels). SCI, spinal cord injury; BMS, Basso Mouse Scale; LSD, least significant difference; GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclear protein; ChAT, choline acetyltransferase; LTG, lamotrigine; Veh, vehicle. Fig. 1a,b were created using Adobe Illustrator (15.0.0, Adobe Systems, San Jose, CA, USA).

Previous research has indicated that LTG attenuates spinal neural injury after SCI. However, the impact of these neuroprotective effects on locomotor function still requires further investigation. We assessed the open-field locomotor function in mice with a complete T9 transection by using a double-blinded BMS scoring system. Initially, both SCI-LTG and SCI-Veh groups exhibited severe impairment in hind limb locomotion, which showed gradual improvement over time. On days 35, 42, 49, 56, and 63 post-injury, BMS scores were significantly higher in LTG-treated mice compared to the Veh group (Fig. 2a,b).

Fig. 2.

Enhanced BMS scores and synapse survival in LTG-treated mice following SCI. (a) Diagram showing motor function recovery in mice following LTG treatment. (b) The timeline displays changes in BMS scores in SCI-Veh and SCI-LTG groups. Data are presented as the mean $\pm$ SEM from 10 independent experiments, with each independent experiment including one mouse per group (n = 10 per group). Two-way ANOVA, Fisher’s LSD. **p 0.01. (c,d) Co-immunolabeling (c) and quantification (d) of vGlut1 and ChAT show surviving synapses between glutamatergic terminals and motor neurons in Control, SCI-Veh, and SCI-LTG groups at 9 weeks after SCI. Glutamatergic synapses per neuron are presented as the mean $\pm$ SEM from 5 independent experiments, each with one mouse per group (n = 5 per group). One-way ANOVA, Bonferroni’s post-test. ****p 0.0001, *p 0.05. The scale bar in the figure is 20 µm. (e,f) Western blot analysis (e) and quantification (f) of vGlut1 expression in Control, SCI-Veh, and SCI-LTG groups. Data shown are the mean $\pm$ SEM from 3 independent experiments, each with 3 mice per group (n = 3 per group). ***p 0.001, *p 0.05, one-way ANOVA, Bonferroni’s post-test. (g) Diagram illustrating spinal neuronal connections after T9 transection. vGlut1, vesicular glutamate transporter 1. Fig. 2a,g were created using BioRender (https://biorender.com/).

Subsequently, we investigated the basis for the improved motor function in LTG-treated mice. Given the enhanced neuronal survival observed in the LTG group (Fig. 1), we hypothesized that synapse survival was also increased, which could partly explain the improved BMS scores. To verify this, we performed co-immunostaining of ChAT and vGlut1 at 9 weeks after SCI. This confirmed the presence of a higher number of glutamatergic synapses in the SCI-LTG group compared with the SCI-Veh group (Fig. 2c,d). Western blot analysis of vGlut1 further supported this finding, indicating more glutamatergic synapses in the LTG-treated group (Fig. 2e,f) (The original western blot data for Fig. 2e are provided in Supplementary Fig. 1). These results suggest that the improvement in BMS scores observed in LTG-treated mice after SCI may be due to enhanced survival of spinal synapses and neurons (Fig. 2g).

We next conducted a series of analyses to elucidate the neuroprotective mechanism of LTG. First, TUNEL staining was performed to detect apoptotic neurons in both the SCI-Veh and SCI-LTG groups. The results showed a reduction in apoptotic neurons in the SCI-LTG group compared to the SCI-Veh group 4 weeks after SCI (Fig. 3a–c). Following this, we conducted western blot analysis of key apoptosis-related proteins, including Bcl-2, Bax, and cleaved caspase-3, 7 days post-injury (Fig. 3d,e) (The original western blot data for Fig. 3e are provided in Supplementary Fig. 2). The SCI-LTG group showed significantly increased Bcl-2 expression levels in comparison to the SCI-Veh group (Fig. 3f), along with reduced expression levels of Bax and cleaved caspase-3 (Fig. 3g,h). These results indicate that LTG effectively suppresses neuronal apoptosis, which may underlie its neuroprotective effects.

Fig. 3.

LTG may reduce neuronal apoptosis in mice after SCI. (a) Diagram illustrating TUNEL staining around the injury site in the mouse spinal cord following SCI. (b,c) TUNEL assay (b) and quantitative analysis (c) of apoptotic neurons in the SCI-Veh and SCI-LTG groups 4 weeks post-injury. The number of TUNEL⁺ neurons was counted, and data are presented as the mean $\pm$ SEM from 5 independent experiments, with each independent experiment including one mouse per group (n = 5 per group). *p 0.05, Student’s t test. Scale bars, 100 µm (left panel), 100 µm (right panel). (d) Diagram illustrating the sampling method from the mouse spinal lesion site for Western blot analysis. (e–h) Western blot analysis (e) and quantification of Bcl-2 (f), Bax (g), and cleaved caspase-3 (h) expression in the lesion site of the Control, SCI-Veh and SCI-LTG groups 7 days post-SCI. Data are presented as the mean $\pm$ SEM from 3 independent experiments, each with 3 mice per group (n = 3 per group). One-way ANOVA, Bonferroni’s post-test. *p 0.05, **p 0.01. Fig. 3a was created using Adobe Illustrator.

To further investigate how LTG suppresses apoptosis and promotes neuronal survival after SCI, we examined whether these effects are linked to the activation of autophagy. We first assessed LC3-II expression as an indicator of autophagosome formation. At 7 days after SCI, a significant increase in LC3-II levels was observed in the SCI-LTG group compared with the SCI-Veh group, indicating enhanced autophagosome formation (Fig. 4a,b) (The original western blot data for Fig. 4a are provided in Supplementary Fig. 3). To determine whether this increase was due to elevated autophagosome production or impaired autolysosome degradation, we examined the expression of p62. This marker accumulates when autolysosome degradation is inhibited. p62 expression was significantly lower in the SCI-LTG group compared to the SCI-Veh group at 7 days post-SCI, suggesting that LTG treatment promotes both autophagosome formation and autolysosome degradation (Fig. 4c). Immunostaining further corroborated these findings, showing reduced p62 and elevated LC3-II levels in the SCI-LTG group compared to the SCI-Veh group (Fig. 4d–f).

Fig. 4.

Enhanced autophagy flux by LTG attenuates neuronal death in SCI. (a–c) Western blot (a) and quantification of LC3-II (b) and p62 (c) expression at the lesion site in Control, SCI-Veh, and SCI-LTG groups 7 days after SCI. Data are presented as the mean $\pm$ SEM of 3 independent experiments, with each experiment including 3 mice per group. (n = 3 per group). *p 0.05, **p 0.01. One-way ANOVA followed by Bonferroni’s post-test. (d–f) Immunostaining (d) and quantification (e,f) of LC3-II and p62 expression in the lesion area among the Control, SCI-Veh, and SCI-LTG groups 7 days after SCI. Data are presented as mean $\pm$ SEM from 5 independent experiments, each with one mouse per group. (n = 5 per group). One-way ANOVA, Bonferroni’s post-test. *p 0.05, **p 0.01. The scale bar in the figure is 100 µm. (g,h) Immunolabeling (g) and quantification (h) of NeuN⁺ neurons in SCI-Veh, SCI-Baf, SCI-LTG and SCI-LTG-Baf groups 4 weeks after SCI. Data shown are the mean $\pm$ SEM from 5 independent experiments, each with one mouse per group (n = 5 per group). One-way ANOVA, Bonferroni’s post-test. **p 0.01. The scale bar in the figure is 200 µm. (i–k) Western blotting (i) and quantitative analysis (j,k) of p62 and LC3-II expression at the lesion area in SCI-Veh, SCI-Baf, SCI-LTG and SCI-LTG-Baf groups 7 days after SCI. Data shown are the mean $\pm$ SEM from 3 independent experiments, each with 3 mice per group. (n = 3 per group). One-way ANOVA, Bonferroni’s post-test. *p 0.05, **p 0.01.

To further elucidate the role of autophagic flux in neuronal survival, we employed Baf, a specific inhibitor of autolysosome degradation [25]. Co-immunostaining for NeuN and GFAP at 4 weeks post-SCI revealed a significant reduction in surviving spinal neurons in the SCI-Baf and SCI-LTG-Baf groups compared with the SCI-Veh and SCI-LTG groups that did not receive Baf treatment. Notably, among the Baf-treated groups, the SCI-LTG-Baf group exhibited a higher neuronal survival rate than the SCI-Baf group (Fig. 4g,h). These findings suggest that Baf treatment decreases the number of surviving neurons by blocking lysosome degradation, thereby exacerbating neuronal death after SCI. LTG mitigates this effect by promoting autophagy flux, which is essential for the survival of spinal neurons.

To assess autophagy-lysosome changes after Baf treatment, p62 and LC3-II were measured at the lesion area 7 days post-SCI. Both the SCI-Baf and SCI-LTG-Baf groups showed increased p62 and LC3-II levels, indicating that Baf effectively suppressed autolysosome degradation in the autophagy-lysosome pathway. In addition, the SCI-LTG-Baf group exhibited greater autolysosome degradation than the SCI-Baf group, suggesting that LTG partially alleviates Baf-induced suppression of autolysosome degradation (Fig. 4i–k) (The original western blot data for Fig. 4i are provided in Supplementary Fig. 4). Elevated autolysosome degradation in the SCI-LTG-Baf group is reflected by the reduced p62 levels, which suggests enhanced autophagic flux. This may explain the reduced neuron loss in LTG-treated mice after SCI and Baf treatment.

In summary, these results demonstrate that autophagy flux is essential for LTG to promote the survival of spinal neurons after SCI by enhancing autophagosome formation and autolysosome degradation.