Experimental study. To investigate the effects of endothelin-receptor antagonist Bosentan on the spinal neural apoptosis in rats with ischemic reperfusion (IR) injury. Department of Neurosurgery, the Second Affiliated Hospital, Xi’an Jiaotong University School of Medcine, Xi’an, Shaanxi Province, China Sprague–Dawley Rats were randomly divided into two groups, saline (IRS, n=48) and Bosentan (IRB, n=48) treatment, respectively, when reperfused in 6 h, 12 h, 24 h, 3 days, 5 days and 7 days. Immunohistochemical staining was used to assess endothelin-1 (ET-1), endothelin receptor type A (ETR_A), endothelin receptor type B (ETR_B), Bcl-2, Bax, Caspase-8, Caspase-9 and Caspase-3 expression. ET-1 and its receptor in spinal cord tissue were evaluated by real-time PCR. Plasma ET-1 concentration was also detected using radioimmunoassay. Compared with the group IRS, plasma concentration of ET-1 in group IRB was significantly increased at each time point (P<0.05) and peaked at 24 h (P<0.01). ETR_B expression in group IRB was significantly higher than group IRS at each time point (P<0.05) and peaked at day 3 (P0.05). The apoptosis rate in group IRB was significantly decreased at each time point (P<0.05). The protein expressions of Bcl-2, Bax, Caspase-8, Caspase-9 and Caspase-3 were significantly increased in response to Bosentan treatment after IR. These results suggest Bosentan decreases apoptosis rate after IR injury in the spinal cord, possibly through the ET-1-ETR_B signaling pathway.

Experimental study. To investigate whether Bosentan, an endothelin-A/-B dual receptor antagonist, could protect neurons after spinal cord ischemia reperfusion (SCIR) injury in rats and its underlying signaling pathway. Department of Neurosurgery, the Second Affiliated Hospital, Xi’an Jiaotong University School of Medicine, Xi’an, Shaanxi Province, China. Sprague–Dawley rats were randomly divided into two groups, saline group (IRS, n=48) and Bosentan group (IRB, 5 mg kg⁻¹, n=48). After ischemia for 1 h with occlusion of the infrarenal aorta, spinal cord were reperfused for 6h, 12h, 24h, 3d, 5d, and 7d separately. Enzyme-linked immunosorbent assay was used to detect vascular endothelial growth factor (VEGF) in serum. Immunohistochemistry was performed to detect protein expression of VEGF, VEGF receptor 1 (FLT-1) and VEGF receptor 2 (FLK-1). Gene expressions of VEGF and its receptors were evaluated using the quantitative reverse transcription polymerase chain reaction. Compared with the IRS group, gene and protein expressions of VEGF, FLT-1 and FLK-1 were significantly increased (P<0.05), so was the concentration of VEGF in plasma (P<0.05). FLK-1 was expressed on spinal cord neurons.

Spinal cord injury (SCI) triggers a complex inflammatory response that impedes neural repair and functional recovery. The modulation of macrophage phenotypes is thus considered a promising therapeutic strategy to mitigate inflammation and promote regeneration. We employed microarray and single-cell RNA sequencing (scRNA-seq) to investigate gene expression changes and immune cell dynamics in mice following crush injury at 3 and 7 days post-injury (dpi). High-dimensional gene co-expression network analysis (hdWGCNA) and slingshot trajectory analysis were employed to identify key gene modules and macrophage differentiation pathways. Subsequently, immunofluorescence staining, flow cytometry, and western blotting were performed to validate the identified effects of amantadine on macrophage differentiation and inflammation. To elucidate the molecular mechanisms underlying the injury response at the transcriptional level, we performed a microarray analysis followed by gene set enrichment analysis (GSEA). The results revealed that pathways related to phagocytosis and macrophage activation are significantly involved post-injury, shedding light on the regulatory role of macrophages in SCI repair. To further investigate macrophage dynamics within the injured spinal cord, we conducted scRNA-Seq, identifying three distinct macrophage subtypes: border-associated macrophages (BAMs), inflammatory macrophages (IMs), and chemotaxis-inducing macrophages (CIMs). Trajectory analysis suggested a differentiation pathway from Il-1b+ IMs to Mrc1+ BAMs, and subsequently to Arg1+ CIMs, indicating a potential maturation process. Given the importance of these pathways in the injury response, we utilized molecular docking to hypothesize that amantadine might modulate this process. Subsequent in vitro and in vivo experiments demonstrated that amantadine reduces Il-1b+ IMs and facilitates the transition to Mrc1+ BAMs and Arg1+ CIMs, likely through modulation of the HIF-1α and NF-κB pathways. This modulation promotes neural regeneration and enhances functional recovery following SCI. Amantadine modulates macrophage phenotypes following SCI, reduces early inflammatory responses, and enhances neural function recovery. These findings highlight the therapeutic potential of amantadine as a treatment for SCI, and provide a foundation for future translational research into its clinical applications.

Deer antler blastema progenitor cells (ABPCs) are promising for regenerative medicine due to their role in annual antler regeneration, the only case of complete organ regeneration in mammals. ABPC-derived signals show great potential for promoting regeneration in tissues with limited natural regenerative ability. Our findings demonstrate the capability of extracellular vesicles from ABPCs (EVs^ABPC) to repair spinal cord injury (SCI), a condition with low regenerative capacity. EVs^ABPC significantly enhanced the proliferation of neural stem cells (NSCs) and activated neuronal regenerative potential, resulting in a 5.2-fold increase in axonal length. Additionally, EVs^ABPC exhibited immunomodulatory effects, shifting macrophages from M1 to M2. Engineered with activated cell-penetrating peptides (ACPPs), EVs^ABPC significantly outperformed EVs from rat bone marrow stem cells (EVs^BMSC) and neural stem cells (EVs^NSC), promoting a 1.3-fold increase in axonal growth, a 30.6% reduction in neuronal apoptosis, and a 2.6-fold improvement in motor function recovery. These findings support ABPC-derived EVs as a promising therapeutic candidate for SCI repair.

Experimental study. To investigate the expression of C5a and its receptor after spinal cord ischemia reperfusion injury (SIRI) in rat. Department of Neurosurgery, the Second Affiliated Hospital, Xi’an Jiaotong University, Shaanxi Province, Xi’an, China Sprague Dawley rats were subjected to 1 h of infrarenal aorta occlusion to induce spinal cord ischemia. Spinal cord was reperfused for 12, 24, 48 h or 3 days separately after ischemia, respectively. Enzyme-linked immunosorbent assay was used to detect C5a in rat serum. Immunofluorescent staining was performed to detect the expression and cellular localization of C5aR in spinal cord following SIRI. Following SIRI, the motor behavior of the rats was significantly compromised. The serum concentration of C5a in the rat was elevated after SIRI and peaked at 24 h. C5aR was significantly upregulated in the lumbar spinal cord following SIRI and expressed on motor neurons and the microglia, but not astrocyte. There was no significant difference in the expression level of C5aR localized on motor neurons after SIRI. Remarkable upregulation of the C5aR may be associated with increased number of C5aR-positive microglia and elevated cellular expression level. This study provides the first evidence of the expression of C5a and its receptors in SIRI and suggests their possible contribution to SIRI in a rat model.